Pest control

ABSTRACT

The present invention relates to novel rodent control agents comprising antibodies, or antigen-binding fragments thereof, that bind to proteins expressed in rodents and in particular antibodies or antigen-binding fragments that bind to proteins expressed in the gastrointestinal (GI) tract of rodents, as well as to methods of making such novel rodent control agents. The invention further extends to novel antibodies and antigen-binding fragments for use in rodent control as well as to methods of controlling rodents through the use of such antibodies, antigen binding fragments and novel rodent control agents.

The present invention relates to novel rodent control agents comprisingantibodies, or antigen-binding fragments thereof, that bind to proteinsexpressed in rodents and in particular antibodies or antigen-bindingfragments that bind to proteins expressed in the gastrointestinal (GI)tract of rodents, as well as to methods of making such novel rodentcontrol agents. The invention further extends to novel antibodies andantigen-binding fragments for use in rodent control as well as tomethods of controlling rodents through the use of such antibodies,antigen binding fragments and novel rodent control agents.

Rodents have long been recognised as pests and cause a variety ofproblems. They frequently scavenge their food from that which isdestined for human and domesticated animal consumption, thus they causedamage to and contaminate (with urine, faeces & disease) not onlygrowing crops, but also stored food materials. Rodents are also known tobe carriers of a wide variety of diseases including for example,Trichinosis, Leptospirosis, Cholera, Bubonic Plague, Typhus, Dysentery,Hantavirus, Salmonellosis, Pasteurellosis, Toxoplasmosis, and Rat bitefever, which may be spread either through direct (e.g. through bites) orindirect (e.g. through dust generated from faeces, urine, and/or saliva,and/or insects which live on or feed off rodents etc.) contact to manand/or other mammalian species. Furthermore, rodents frequently causephysical damage to property, installations and equipment through gnawingand burrowing.

Methods of controlling rodents have thus evolved over the years andthese can in general be split into three categories: i) trapping; ii)killing through exposure to chemical agents; and iii) sterilising orreducing the ability of rodents to breed. All three of thesemethodologies suffer from one or more deficiencies.

For example, mechanical traps that are designed to catch and/or killrodents are by their nature limited in their use by the number ofanimals that they can deal with i.e. they may only be able to deal withone animal at a time before they require human intervention to be reset.When one considers the high rate at which rodents can reproduce (with anaverage litter size of 6-8 and between 10 and 12 litters per year, asingle pair of rats can produce as many as 15 000 descendants in ayear), it can be seen that trapping is not suitable for largeinfestations. Furthermore trapping is relatively labour-intensive,requiring regular human intervention and this leads not only toincreased cost with pest control but can also frighten away the rodentsthat are targeted by the traps.

The use of chemical rodenticides is currently the main method forpractical rodent control programmes in both urban and agriculturalenvironments. In general chemical rodenticides are either acute orchronic in their action, i.e. the chemical mediates toxicosis eitherrapidly or slowly after an effective dose has been ingested by therodent. Acute rodenticides include zinc phosphide, trizinc phosphide,red squill (active ingredient scilliroside), sodium (mono)fluoroacetate,fluoroacetamide, alphachloralose, and thallium sulphate. Somerodenticidal chemicals, such as calciferol, bromethalin andflupropadine, may be described as sub-acute rodenticides in that alethal dose is ingested in the first 24 hours, but repeated feedingoccurs and death is normally delayed for several days, however thedistinction between acute and sub-acute acting rodenticides is notalways clear. Whilst acute rodenticides are advantageous in that theyact very rapidly, they are in general very toxic chemicals and there aresafety and environmental concerns associated with their use.Furthermore, survivors of acute poison baiting may become bait-shy, thusreducing the overall efficacy of this method of rodent control.

Chronic rodenticides mediate their effect by acting as anti-coagulants,examples of which include the first generation anticoagulantshydroxycoumarins (e.g. warfarin, coumachlor, coumafuryl, coumatetralyl)and indane-diones (e.g. pindone, diphacinone, chlorphacinone); and thesecond generation anti-coagulants bromadiolone, brodifacoum, difenacoum,flocoumafen, and difethialone. The use of second generation rodenticideshas been widespread over the last 2 to 3 decades, and even longer (3 to5 decades) for the first generation anti-coagulants. Thus rodentpopulations have had ample time to develop resistance to this method ofcontrol, and resistance/tolerance has been reported to each of theactive ingredients mentioned above in various rodent populations and/orspecies (see Kerrins et al., 2001, “Distribution of resistances toanticoagulant rodenticides in England, 1995-98” pp 149-159 Proceedings,Advances in Vertebrate Pest Management II, Second European VertebratePest Management Conference, Braunschweig, Germany).

A further disadvantage of second-generation anti-coagulants stems fromtheir non-species specific mode of action coupled to their widespreaduse. For many years there have been concerns over secondary poisoning ofpredatory and scavenging mammals and birds that consume rodents carryinganti-coagulant residues. Both brodifacoum and flocoumafen have beenrestricted to indoor use in the United Kingdom as they are thought topose an unacceptably high risk of secondary poisoning if used outside.However, widespread resistance to the more widely used difenacoum andbromadiolone may encourage misuse of the more potent anti-coagulants theuse of which is restricted to indoors. More recently low-levels ofresidues and a lethal impact of such residues have been observed in awide range of non-target species that are of considerable conservationimportance e.g. Barn Owls, Stoats, Weasels, Polecats and Kestrels.

A third method of rodent control that has been proposed, but which hasnot as yet achieved any real commercial success, relies on reducingrodent birth rate. The use of reproductive inhibitors, such ascontraceptives and gametocides, as well as the use of biological andchemical sterilants have all been investigated. However, each approachsuffers from some disadvantage. For example, whilst the use of chemicalor steroidal compounds as anti-fertility agents has proved successfulfor captive animals, they are more-difficult to employ with free-rangingpest populations. The compounds are unpalatable and thus it is difficultto ensure that rodents obtain an adequate dose. The gametocidealphachlorohydrin has been marketed as a toxicant-sterilant, however, ithas variable effects in different species and is toxic (leading to up to50% mortality) at higher doses. More recent research has examinedimmunocontraception as a method of rodent control (see for example, USPatent Application No 2005/0009188; Moore & Wong 1997, ReproductionFertility & Development 9:125-9; Smith et al., 1997, Reproduction,Fertility & Development, 9:85-9). However, there are difficulties withefficacy when route of administration is oral, due to problems of oraltolerance, and adjuvants may be required.

There is thus a need for novel rodent control agents (i.e. agents thatare capable of controlling a population of rodents, for example throughkilling rodents or by regulating the ability of a population of rodentsto breed), which overcome some of the disadvantages suffered by currentmethods of rodent control. The present invention addresses this need byproviding novel rodent control agents that comprise an antibodycomponent, which binds to an extracellular epitope of a protein that isexpressed in rodents (such a protein is referred to herein as a targetprotein). Thus in a first aspect of the invention there is provided arodent control agent comprising an antibody component that binds to anextracellular epitope of a protein that is expressed in a rodent.

By targeting the rodent control agent to a specific protein and/orspecific tissue in a rodent, for example to a protein expressed in therodent gut tissue/the rodent GI tract, the length of time that the novelrodent control agent is in contact with rodent gut is increased relativeto that of non-specific rodent control agents, which merely pass throughthe gut and are not retarded through specific binding. This may in turnlead to an effective increase in potency of the novel rodent controlagent, thus enabling it to be used at lower concentrations thantraditional non-specific rodenticides, such as anti-coagulantrodenticides.

Preferably the antibody component confers on the novel rodent controlagent selectivity for rodents over non-target animals (e.g. humans,birds, companion animals, farm animals, and wild-animals that are notpests). This selectivity for rodent tissue/rodent protein overnon-target species is further advantageous as it is likely to reduce theneed for antidotes and has the potential to reduce the environmentalimpact of the novel rodent control agent on non-target species.

Where the antibody component recognises a target protein that performs anon-essential function in the rodent, it will be necessary for therodent control agent to further comprise a toxic component or acontraceptive component.

Thus in one embodiment the invention provides a rodent control agentcomprising an antibody component linked to either a toxic component or acontraceptive component. Such novel rodent control agents may be in theform of a fusion protein, wherein the toxic component or contraceptivecomponent is a protein or peptide moiety that is linked either directlyor indirectly (i.e. via a peptide linker) to the antibody component viaa peptide bond, or in the form of a protein conjugate, wherein the toxicor contraceptive component is either a small molecule (i.e. anon-proteinaceous, chemical, entity) or protein or peptide moiety thatis directly chemically conjugated to the antibody component.

Where the antibody component recognises a target protein that performsan essential function in the rodent (for example, a protein thatperforms an essential function in the GI tract of a rodent), theantibody component may achieve the rodent control function as the solefunctional agent. Such a rodent control agent would mediate its effectby virtue of the antibody component binding to the essential targetprotein and thus blocking or inhibiting the function of the essentialprotein. Thus in a further embodiment the present invention provides arodent control agent comprising an antibody component that binds to anextracellular epitope of a protein that is expressed in a rodent,wherein the protein performs an essential function in the rodent.Examples of suitable target proteins against which antibody componentsaccording to this aspect of the invention bind (and thus which may beused in producing antibody components according to this aspect of theinvention) include a rodent Sox10 gene product, a rodent endothelinreceptor (EDNRB), a rodent endothelin-3 ligand (EDN3), a rodent CFTR(see Table 1 below and the Examples for more detail), rodent IL-2,rodent IL-10, rodent T-cell receptor alpha and/or beta chains, rodentcomponents of the class II major histocompatibility complex. The skilledman will appreciate that some of the above-mentioned geneproducts/proteins are not readily accessible on an epithelial surface ofa rodent and thus require internalisation before they are capable ofmediating their activity. In such a case, it may be desirable to combinean antibody component directed to one of the above proteins with afurther targeting antibody component, which enhances the probability ofinternalisation. Thus two or more antibody components, one directedagainst one of the afore-mentioned proteins and a second acting astargeting antibody to a suitable epithelial target to enhanceinternalisation, may be linked, either via conjugation or by virtue of afusion protein as described hereinafter.

The term “antibody component” is used herein to mean an antibody orantigen-binding fragment thereof, which binds to an extracellularepitope of a protein that is expressed in a rodent. Preferably theepitope to which the antibody component binds has an amino acid sequencethat is only found in a rodent protein, i.e. the antibody binds to arodent specific epitope or RSE. Rodent specific epitopes (RSEs), whichmay be used in the generation of antibody components for use in rodentcontrol agents of the invention and to which rodent control agent of theinvention bind, form a second aspect of the invention described herein.Antibody components which bind to RSEs are considered as a third aspectof the invention described herein.

The RSE will be extracellular to facilitate access of the antibody (orantigen-binding fragment) to its binding site. Preferably the targetprotein providing the RSE will be expressed in or on an epithelialsurface of the rodent including, for example, the epithelia of the nose,mouth, eyes, gastro-intestinal tract, genito-urinary tract andepidermis. Most preferably the protein will be expressed in (or on) thegastro-intestinal epithelium of a rodent.

In one embodiment the RSE will be provided by a continuous (i.e.sequential) peptide sequence (i.e. the first amino acid residue of theepitope will be directly linked via a peptide bond to the second aminoacid residue of the epitope, the second amino acid residue of theepitope will be linked via a peptide bond to the third amino acidresidue, etc.). Such a continuous peptide epitope is referred to hereinas a rodent-specific peptide epitope or RSPE. RSPEs to which antibodycomponents of the invention bind, and which may thus be used ingenerating antibody components (and also rodent control agents) of theinvention, may be determined by comparative bioinformatic analysis ofproteins that are expressed in the desired target tissue/cell layer asindicated by literature and database information, followed byconfirmatory immunological analysis. RSPEs, like RSEs, are found innature only in rodent proteins. An RSPE is defined herein as anoligopeptide fragment of a target protein, wherein the oligopeptidesequence represents an extracellular continuous peptide epitope that hasa percentage identity of 60% or less with a corresponding linear peptidesequence from a homologous protein from a non-target (i.e. non-rodent,for example, human) animal.

The term “oligopeptide fragment” as used herein refers to a fragment ofa target protein, said fragment consisting of at least about 4 and atmost about 50 amino acids. Preferably said oligopeptide fragment will bebetween 9 and 45 (inclusive) amino acids in length and more preferablysaid oligopeptide fragment will be between 9 to 30 (inclusive) aminoacids in length. In specific embodiments said oligopeptide fragment willbe 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24 or 44 amino acids inlength.

Preferably the percentage of identity between the RSPE and thenon-target protein will be such that an antibody component of theinvention has high specificity and affinity for the RSPE and will notcross-react with a non-rodent protein of the same class/family as thatfrom which the RSPE was derived. In preferred embodiments the percentageidentity between the RSPE and the non-target protein, and in particularbetween the RSPE and a corresponding linear (i.e. continuous orsequential) peptide sequence from a homologous protein from a non-targetanimal, is less than or equal to 50%, 45%, 40%, 35%, 30%, or 25%.

Preferably RSPEs will be highly conserved between all target rodentspecies, in particular highly conserved between rats and mice,especially between two or more of Rattus rattus, Rattus norvegicus andMus musculus/Mus domesticus. By highly conserved it is meant that thepercentage identity between a RSPE from one rodent species and thecorresponding RSPE from a second rodent species will be high. Preferablythe percentage identity between two rodent RSPEs will be at least 75%.More preferably the percentage identity will be greater than or equal to80%, 85%, 90% or 95%, 96%, 97%, 98%, or 99%. Most preferably the RSPEwill be 100% identical between two or more rodent species.

Although it is preferred that the percentage identity between of an RSPEis highly conserved between two or more species as discussed above, ifthe desired high level of conservation is not observed, different RPSEsmay be used from proteins (either homologous or different) from two ormore rodent species, to generate different antibody components, whichmay then be combined in a rodent control agent as described hereinafter(e.g. each antibody component linked to a toxic/contraceptive componentwith the rodent control agent thus comprising multiple differentantibody-toxin/contraceptive molecules or the rodent control agentcomprising a toxic/contraceptive component linked to two or moredifferent antibody components, each of the two or more antibodycomponents recognising an RSPE from a protein from a different rodentspecies). In one specific embodiment, an antibody component recognisingmouse MDR1 (SEQ ID NO: 8) could be combined with an antibody componentrecognising rat MDR1 (SEQ ID NO: 9), in rodent control agents of theinvention.

Examples of target proteins to which antibody components of theinvention bind, and which may be used in producing or identifyingantibody components of the invention, are given in Table 1. This Tablealso provides examples of specific RSPEs which may be derived from theexemplary proteins. The skilled man will appreciate that neither thelist of proteins given in Table 1, nor the list of RSPEs derivedtherefrom, is exhaustive. Further suitable RSPEs may be identifiedwithin the given proteins (all of which are expressed in the GI tract),and further proteins, for example from other rodent target tissues suchas the nasal epithelia, buccal epithelia, epidermis, epithelia of thegenito-urinary tract and epithelia of the eye, may also be used toproduce antibody components of the invention.

TABLE 1 Examples of Proteins which may be targeted by antibodies of thepresent invention, and specific examples of RSPEs which may be derivedfrom such proteins. SwissProt/ GenPept RSPE primary Accession amino acidSource Protein Number sequence SEQ ID NO Rat oligopeptide P51574VIRSRASDGCLEVKE SEQ ID NO: 1 transporter PepT1 Rat oligopeptide P51574CSSDFKSSNLD SEQ ID NO: 2 transporter PepT1 Rat CD155 (PVR, Q9R1E1SNVNGSYREMKETGSQP SEQ ID NO: 3 Tage4) Rat GTR2 (GLUT2) P12336GTDTPLIVTPAHTTP SEQ ID NO: 4 glucose transporter Rat CFTR chlorideP34158 LKNNPVNGGNNGTKIA SEQ ID NO: 5 transporter Rat CNT2 nucleosideQ62773/ WQDKESSLRNLAK SEQ ID NO: 6 transporter Q9QWI3 Rat CATB(0+)XP233305 GGDMFMNISWVN SEQ ID NO: 7 (slc6a14) colonic amino acidtransporter Rat CATB(0+) XP233305 DTGGDMFMNISWVNS SEQ ID NO: 36(slc6a14) colonic amino acid transporter Rat MDR1 P43245 SFTPSRDPHSDRAITSEQ ID NO: 8 multidrug resistance transporter Mouse MDR1 P06795SFTKAEASILPSIT SEQ ID NO: 9 multidrug resistance transporter RatSucrase- P23739 YNAESITNENAGLKATL SEQ ID NO: 10 Isomaltase Mouse GLUT7glucose XP487836 NTPHKVLKSFYN SEQ ID NO: 11 transporter Mouse GLUT7/RatXP487836/ YYDRNKENIES SEQ ID NO: 12 GTR5 (GLUT5) P43427 glucosetransporters Rat Npt2a (slc34a1) Q06496 PETKEASTSMSRVEA SEQ ID NO: 13sodium/phosphate transporter Rat OATP-B Q9JHI3 LGAQPGPSLFPGCSEPCS SEQ IDNO: 14 (SLC21A9) organic CQSDDF anion transporting polypeptide RatOATP-B Q9JHI3 QPGPSLFPGCSEPCSCQ SEQ ID NO: 15 (SLC21A9) organic aniontransporting polypeptide Rat ASBT apical Q62633 DAEFLEKTDNDMD SEQ ID NO:16 sodium-dependent bile transporter Rat CaT1 (ECAC2) Q9R186QAFQQQDDLYSE SEQ ID NO: 17 calcium transporter Rat OATP3 organic O88397/SYKGVQHQLHVESKVL SEQ ID NO: 18 anion transporting Q9EQR8 polypeptide RatABCG8 sterol P58428/ QIQFNGHIYTTQIG SEQ ID NO: 19 transporter Q8CIQ5/Q923R7 Rat GTR8 (GLUT8) Q9JJZ1/ HVGLLVPISAEPADVHLG SEQ ID NO: 20 glucosetransporter Q9JMA6 Rat MRP1 multidrug Q810E4/ MFAGPEILELIINF SEQ ID NO:21 resistance associated Q8CG09/ protein/ABC Q810G9 transporter Rat CNT1sodium- Q62674 HSHSSLPEGEGGLNKA SEQ ID NO: 22 nucleoside cotransporterRat UT-B urea P70633/ PSKLFMPVSSVP SEQ ID NO: 23 transporter P97689 RatDRA1 chloride/ Q924C9 LSSSSAENDSMIEEKVMV SEQ ID NO: 24 anion exchangerMouse ENT1 Q9JIM1/ KARHCGAQRHHFVFKH SEQ ID NO: 25 equilibrative Q99K84/nucleoside Q9DBT8/ transporter Q9JHF0 Rat ENT1 O54698 TNQSCESTEALADPSVSLSEQ ID NO: 26 equilibrative nucleoside transporter Rat GCC (guanylylP23897 VSGRFPSERS SEQ ID NO: 27 cyclase) Rat PLB O54728AEDLWIQAKELVRHLKDN SEQ ID NO: 28 (phospholipase B) P Rat LPH (lactase-Q02401/ EDAAPTASPVQS SEQ ID NO: 29 phlorizin hydrolase) Q63712/ Q63719Mouse LPH (lactase- XP129479 RYVQVCALCRFSTVFSPR SEQ ID NO 30 phlorizinhydrolase) LPEPVKGERRFSHISLNQ DLPRPLFP Rat AMPN P15684/ GSTSATTSTTNPASEQ ID NO: 31 (aminopeptidase N) Q9JHP4 Rat MCDL (mucin and Q9JIK1NKDILLTTVPMETERTIR SEQ ID NO: 32 cadherin-like protein) Rat SCAB(amiloride- P37090/ LPQDLVGMGYAPDRI SEQ ID NO: 33 sensitive sodiumO09183 channel beta-subunit) Rat SCAB (amiloride- P37090/ SSNPAPGST SEQID NO: 34 sensitive sodium O09183 channel beta-subunit) Rat KCV2(potassium XP220024 DQRHGKGSPREHDLE SEQ ID NO: 35 voltage-gated channelsubfamily V member 2)

Examples of preferred target proteins for use in the invention are therat oligopeptide transporter PepT1, rat CD155, rat GTR2, rat CFTR, ratCNT2, rat CATB(0)+, rat MDR1, mouse MDR1, rat sucrase-isomaltase, mouseGLUT7, rat OATP-B, rat ENT1, rat GCC, rat PLB, rat LPH, rat AMPN, ratMCDL, and rat SCAB.

Preferred RSPEs of the invention have SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 26, 27, 28, 29, 31, 32 and 34.

Particularly preferred target proteins for use in the invention are ratoligopeptide transporter PepT1, rat CD155, rat CFTR, rat CNT2, mouseGLUT7, rat GCC, rat PLB and rat LPH.

Particularly preferred RSPEs of the invention have SEQ ID NOs 1, 3, 5,6, 11, and 27, 28 and 29.

The skilled man will appreciate that antibodies recognise tertiaryprotein structure and that an epitope may comprise amino acid residuesthat are distributed throughout the primary amino acid sequence of aprotein whilst being structurally in close proximity to each other. Thusin a further embodiment antibody components (and thus also rodentcontrol agents) of the invention may recognise a discontinuousextracellular RSE.

Antibodies for use in the invention may be polyclonal or monoclonal.Such antibodies may be obtained by immunising an animal with a RSPE asdescribed above, or by immunising an animal with the intact rodenttarget protein. Polyclonal antibodies of the invention may be obtainedfrom the serum of such an immunised animal, see for example, Example 2.Polyclonal antibodies which recognise the RSPE or rodent target proteinbut which do not recognise a homologous protein from a non-target animalmay be identified using standard immunological techniques (for exampleby ELISA, and/or through immunohistochemistry against appropriate tissuesources).

Monoclonal antibodies for use in the invention may be obtained byisolating B-lymphocytes from the spleen of an animal immunised with anRSPE or rodent target protein and making a hybridoma cell-line with suchlymphocytes. The hybridoma cell-lines are then screened for those whichsecrete antibodies that recognise the RSPE or rodent target protein butwhich do not recognise a homologous protein from a non-target animal,see for example, the Examples.

Preferred polyclonal and monoclonal, antibodies for use in the inventionare described herein in the Examples.

Antibodies and antigen-binding fragments for use in the invention mayalso be isolated from a bacteriophage display library (naive, or immune)of antibodies or antigen-binding fragments, or from a similar yeast orbacterial display library of antibodies or antigen-binding fragments, orfrom a ribosomal display library of antibodies or antigen-bindingfragments. Typically such display libraries will be screened to identifydisplayed proteins that bind to RSPEs or rodent target proteins. Theskilled man will be familiar with the methodology for screening suchlibraries, identifying binding-partners in the library and subsequentlyisolating such members. Antibody and antigen-binding fragment displaylibraries which may be used for screening, and their construction aredescribed in the art (see for example: International Patent PublicationNos WO 01/90190 & WO 93/19172; U.S. Pat. Nos. 5,759,808 & 6,517,829;review by Hoogenboom 1997, Trends in Biotechnology 15(2):62-70; Dooleyet al., 2003 Molecular Immunology 40: 25-33; Nutall et al., 2001Molecular Immunology 2001, 38:313-326; and Hanes et al., 1998Proceedings of the National Academy of Sciences USA 95: 14130-14135).Again antibodies or antigen-binding fragments identified in this waywill be checked to confirm that they do not bind to a homologous proteinfrom a non-target animal.

Antibodies for use in the invention may be of any immunoglobulin class,e.g. they may be an IgA, IgD, IgE, IgG or IgM antibody. Preferably anantibody of the invention will be an IgG antibody. Antibodies of theinvention may be immunoglobulin molecules comprising both heavy andlight chains, or they may be single-chain antibodies. The term“single-chain antibody” as used herein encompasses both naturallyoccurring antibodies which consist of only a single-type of chain e.g.Camelid or Chondricthyes (shark) derived antibodies, which are devoid oflight chains, as well as engineered antibodies that consist of only asingle polypeptide chain. Examples of such engineered antibodies includefor example, single chain antibodies or “minibodies” as described inInternational Patent Publication No. WO94/09817 and single-chainvariable fragments (scFv). Antibodies of the invention are preferablysingle chain antibodies. In one preferred embodiment the antibody is ascFv. In another preferred embodiment the antibody is a single-chainantibody devoid of light chains, most preferably a Camelid derivedantibody (for example as described in International Patent PublicationNo. WO 94/04678).

Antigen-binding fragments for use in the invention includeimmunoglobulin light chains, immunoglobulin heavy chains, V_(H) domains,V_(L) domains, Fvs, Fabs, di-Fabs, Fab′s, F(ab′)₂s, V_(HH) domains,IgNAR V domains and CDRs. The skilled man will be very familiar withantigen-binding fragments such as immunoglobulin light and heavy chains,V_(H) and V_(L) domains, Fvs, Fabs, di-Fabs, Fab′s, F(ab′)₂s, and CDRs,and their preparation. AV_(HH) domain is the variable domain of aCamelid antibody. V_(HH) domains and their isolation are described inthe art, see for example International Patent Publication No. WO94/04678, International Patent Publication No. WO 01/90190 and thereferences contained therein. IgNAR antibodies are single-chainantibodies from sharks, which, in common with the Camelid antibodies,are devoid of light chains (see Greenberg et al., 1995 Nature374:168-173). The antigen-binding region of these antibodies, the IgNARvariable domain (IgNAR V domain), has also been described in the art,see for example Dooley et al., 2003 (Molecular Immunology 40:25-33) andreferences cited therein. In some embodiments, the antibody componentfor use in the invention will be a Camelid antibody, or a V_(HH) domain,which recognises one of the preferred target proteins describedhereinbefore. In particular, Camelid antibodies or V_(HH) domains whichbind to any one of SEQ ID NOs 1, 3, 5, 6, 11, or 27 are preferred,whilst V_(HH) domains binding to any one of SEQ ID NOs 5, 6, 11 or 27are particularly preferred.

Antibodies and antigen-binding fragments used in the invention may alsobe engineered to increase their stability, for example they may bestabilised by disulfide bridges (see for example Reiter et al., 1996,Nature Biotechnology 14(10):1239-1245).

As described above, antibody components may be obtained by using intactrodent proteins or RSEs (which term includes RSPEs), either as antigensor to screen for suitable antibodies/antigen-binding fragments, whichhave been selected as having a low percentage identity to homologousproteins from non-target (i.e. non-rodent, for example, human) animals.The probability of an antibody component of the present inventioncross-reacting with a homologous protein from a non-target species isthus reduced. Thus, in preferred embodiments of the invention, antibodycomponents (and thus also rodent control agents of the invention)exhibit selectivity for an epitope on a rodent target protein ratherthan the corresponding epitope on a homologous protein from a non-targetanimal. In other words preferred antibody components of the inventionshow selectivity to a RSE by binding thereto (or to the rodent proteinfrom which the RSE is derived) with a greater affinity than to acorresponding epitope on a homologous protein (or the homologousprotein) from a non-target animal. Non-target animals include humans,birds, companion animals, farm animals, and wild-animals that are notpests.

Preferably antibody components of the invention will exhibit reducedbinding to, and even more preferably not bind, to homologous proteinsfrom humans. Even more preferably antibody components of the inventionwill not only exhibit reduced binding (or not bind) to homologousproteins from humans, but will also exhibit reduced binding (or notbind) to homologous proteins from at least one other non-target animal.

For the avoidance of doubt, the term “bind” insofar is used herein todescribe the interaction of an antibody component of the invention withan epitope or protein in a qualitative or quantitative manner.

Where the term is used qualitatively, specificity for binding to atarget protein, RSE or RSPE may be demonstrated by the ability of theantibody component to bind to the target protein, RSE, or RSPE in adisplaceable manner, wherein displacement of antibody binding resultsfrom the presence of the antigen to which the antibody component wasraised or screened (referred to hereinafter as the “specific antigen”).If binding of such a target-protein/RSE/RSPE-specific antibody componentto a non-target protein (or a corresponding epitope from a non-targetprotein) is not observed, or if some binding to a non-target protein (orcorresponding epitope from a non-target protein) is observed which isnot displaceable by the specific antigen, then the antibody componentwill be deemed as showing selectivity for the target protein, RSE orRSPE (as appropriate) in that the antibody component binds to the targetprotein/RSE/RSPE with a greater affinity than to a non-target protein orcorresponding epitope from a non-target protein. Specificity andselectivity may thus be determined through immunohistochemical analysisof appropriate tissue samples, and/or through ELISA with appropriatesamples.

Where the term “bind” is used quantitatively, this means that theantibody component has an affinity of at least 10⁻⁶ M for the epitope orprotein with which it interacts. Preferably the affinity will be atleast 10⁻⁷M, more preferably 10⁻⁸M, more preferably 10⁻⁹M, even morepreferably 10⁻¹⁰M and most preferably 10⁻¹¹M or greater. Thus where anantibody component binds to an RSE or RSPE (or the protein from whichthe RSE or RSPE is derived) it will have an affinity of at least 10⁻⁶M,and in further embodiments it will have an affinity of at least 10⁻⁷M,at least 10⁻⁸M, at least 10⁻⁹M and at least 10⁻¹⁰M. In preferredembodiments the antibody component will either not bind to acorresponding epitope on a homologous protein from one or morenon-target species (preferably human) i.e. the affinity of the antibodycomponent for a homologous protein from a non-target species will beless than 10⁻⁶M, or it will exhibit reduced binding thereto i.e. theaffinity of the antibody component for a homologous protein from anon-target species will be at least 10-fold less than for the targetrodent protein. Thus, in preferred embodiments, where an antibodycomponent of the invention has an affinity of at least 10⁻⁶M for an RSEor RSPE (or the protein from which the RSE or RSPE is derived), theaffinity of the antibody component for a homologous protein from anon-target species will be less than 10⁻⁶M, and preferably 10⁻⁵M orlower; where an antibody component of the invention has an affinity ofat least 10⁻⁷M for an RSE or RSPE (or the protein from which the RSE orRSPE is derived), the affinity of the antibody component for ahomologous protein from a non-target species will be 10⁻⁶M or lower;where an antibody component of the invention has an affinity of at least10⁻⁸M for an RSE or RSPE (or the protein from which the RSE or RSPE isderived), the affinity of the antibody component for a homologousprotein from a non-target species will be 10⁻⁷M or less, and preferably10⁻⁶M or lower; where an antibody component of the invention has anaffinity of at least 10⁻⁹M for an RSE or RSPE (or the protein from whichthe RSE or RSPE is derived), the affinity of the antibody component fora homologous protein from a non-target species will be 10⁻⁸M or lower,and preferably 10⁻⁶M or lower.

The affinity of the antibody component to target and non-target proteins(as well as to epitopes therefrom) may be determined using anyappropriate technique; for example, through the use of surface plasmonresonance (e.g. with BIAcore™).

In one embodiment of the present invention the rodent control agent isin the form of a fusion protein, wherein the fusion protein comprises afirst protein component and a second protein component, said firstprotein component is an antibody component as described hereinbefore,and said second protein component is selected from the group consistingof a toxin, an immunogen and a hormone.

The first protein component may be linked directly to the second proteincomponent, however, it is preferred that the two components will beindirectly linked through a linker peptide. The linker peptide will ingeneral be of a length that is sufficient for the first component andsecond components to function as desired without one component adverselyeffecting the function of the other (for example by steric hindrance).An example of a commonly used linker peptide, suitable for use in thisaspect of the invention is the (Gly₄Ser)_(n) linker, where “n” is aninteger greater than or equal to 1. Typically n is greater than or equalto 3. Preferably, the primary sequence of the linker peptide is designedto be stable in harsh hydrolytic and thermal environments. This may beachieved by removing or mutating residues in the linker that act asrecognition sites for processing of the linker via, for example, aproteolytic mechanism. Further examples of suitable linker peptides arethose described by Gustavsson et al., 2001 (Protein Engineering14:711-715), Hennecke et al., 1998 (Protein Engineering 11:405-410) andHuston et al., 1991 (Methods in Enzymology 203:46-88).

In a further embodiment, it is desirable to engineer specificinstability into the linker peptide such that controlled separation ofthe two protein components/release of the second protein component iseffected upon delivery of the fusion protein to an appropriate locus,e.g. once the fusion protein has been internalised the second proteincomponent may be released intracellularly. The described control ofseparation may serve to enhance the activity of certain fusion proteins.

As mentioned above, in one embodiment of the fusion protein, the secondprotein component is a toxin. The toxin confers on the fusion protein arodenticidal activity: it effects toxicity against a targeted rodentcell via either an externally- or internally-mediated mode-of-action.Suitable toxins for use in this aspect of the invention include interalia proteins which disrupt membranes, ribosyltransferases, serineproteases, guanylyl cylase activators, proteins involved in ATP-asemediated ion transport, calmodulin-dependent adenylyl cyclases, RNAglycosidases and ribonucleases. Specific examples of suitable toxins aregiven below in Table 2. The skilled man will appreciate some of thetoxins listed in Table 2 are representative of a family of toxinmolecules and where this is the case, any one of those members may alsobe used in the invention.

TABLE 2 Examples of proteins which may be used as toxins in fusionproteins of the invention. SwissProt/ UniProt EMBL Accession AccessionProtein Mode-of-Action Number No Perfringolysin O (theta toxin)cholesterol-dependent pore- P19995 M81080 forming cytolysinAlpha-haemolysin (Hla) pore-forming cytolysin P09616 X01645Sphingomyelinase (beta toxin) phospholipase C/membrane P09978 X61716disrupting Delta-haemolysin cationic amphipathic pore- P01506 AF230358forming lytic peptide Granzyme B serine protease/inducer of P04187/X04072/ apoptosis P18291 M34097 Alpha toxin phospholipase C/membraneP15310 X17300 disrupting Cyt toxin pore-forming/membrane Q04470 Z14147e.g. Cyt 2a disrupting Diphtheria toxin NAD(+)-diphthamide ADP- P00588X00703 ribosyltransferase Granulysin pore-forming/membrane P22749 X54101disrupting Melittin cationic amphipathic pore- P01501 X02007 forminglytic peptide Perforin Calcium-binding pore- P14222 M31951 formingcytolysin Cholera enterotoxin NAD(+) ADP- P01555/ X00171ribosyltransferase P01556 Heat-stable enterotoxin guanylyl cyclaseactivator Q47185 M18345 Equinatoxin pore-forming cytolysin P61914 U41661Listeriolysin cholesterol-dependent pore- P13128 X15127 formingcytolysin VIP2 NAD(+) ADP- Q844J9 AY245547 ribosyltransferase Accessoryenterotoxin ATPase-mediated ion P38441 Z22569 transport Aerolysincholesterol-dependent pore- P09167 M16495 forming cytolysin BinA/BinBpore-forming cytolysins P06575/ Y00378/ P10565 X07992 Colicin E1transmembrane P02978 J01563 depolarisation Haemolysin A pore-formingcytolysin P08715 M14107 CTX IV protein kinase C inhibitor P01443 Y12491Type 2 Ribosome- rRNA N-glycosidase P02879 X03179 inactivating proteins,e.g. Ricin Amoebapore pore-forming peptide P34095 M83945 El Torhaemolysin pore-forming/membrane- P09545 Y00557 disrupting cytolysinVibrio damsela phospholipase D/membrane Q60079 L16584 Haemolysindisrupting Pneumolysin cholesterol-dependent pore- P11990 X52474 formingcytolysin Streptolysin O cholesterol-dependent pore- P21131 M18638forming cytolysin thermostable direct pore-forming cytolysin P19249D90101 haemolysin (Kanagawa toxin) Leptospira haemolysinpore-forming/membrane O34095 U89708 disrupting Cry toxin e.g. Cry1Acpore-forming cytolysin P05068 M11068 Anthrax toxin proteolytic attack ofkinases P15917/ M29081/ in target cell/calmodulin- P13423/ M22589/dependent adenylyl cyclase P40136 M23179 Pseudomonas exotoxin ANAD-dependent ADP- P11439 K01397 ribosyltransferase Barnase ribonucleaseP00648 M14442 VIP3 pore-forming cytolysin Q45792 L48811 ThioninCytolytic plant toxin P01543 AF004018 Type 1 Ribosome- rRNAN-glycosidase P33186 L12243 inactivating proteins e.g. GeloninBeta-purothionin Cytolytic plant toxin P01543 AF004018

Preferred toxins for use in embodiments of the invention includegranzyme B, cyt 2A, β-purothionin, VIP2A, gelonin, and granulysin.Particularly preferred toxins are selected from the group consisting ofgranzyme B, cyt2A, β-purothionin, VIP2A and gelonin.

Protein toxins as described above, may be incorporated in their entiretyin fusion proteins of invention. Alternatively, where a domain orfragment of such a toxin confers the toxic activity, that domain orfragment may be employed as the second protein component in the fusionprotein.

In another embodiment the second protein component in a fusion proteinof the invention is an immunogen, i.e. a poly- or oligo-peptide that iscapable of eliciting an immune response in rodents. In a preferredembodiment, immunogen-fusion proteins of the invention will be capableof acting as immunocontraceptives and will thus act as rodent controlagents by preventing reproduction. Sperm or ovum specific antigens, suchas for example, lactate dehydrogenase C, the sperm antigen PH-20(Primakoff et al., 1988 Nature 335:543-6), fertilin (PH-30) and zonapellucida antigens are thus suitable immunogens for use as the secondprotein component in fusion proteins of the invention.

In yet another embodiment, the second protein component in a fusionprotein of the invention is a hormone or proteinaceous hormone mimetic.In this embodiment, the second protein component will interfere with andprevent reproduction in rodents and the fusion protein thus acts as arodent control agent through the prevention of breeding. An example of ahormone that may be used in this embodiment of the invention includesgonadotrophin releasing hormone.

In further embodiments, fusion proteins as described above may compriseat least one further protein component. This further protein component(or components) may be a toxin, immunogen, hormone or proteinaceoushormone mimetic as described above. Accordingly a fusion protein may beconstructed comprising an antibody component and at least two furtherprotein components, wherein each of the further protein componentsconfers a rodent control (i.e. toxic or contraceptive, or both) functionon the fusion protein. The second and further protein component orcomponents may each have the same, or different rodent control functions(i.e. they may each independently be toxic or contraceptive), and wherethey have the same function they may each have the same, orindependently different, mode of action. Where the additional proteincomponent(s) have the same rodent control function and wherein at leastone additional protein component has a different mode of action to thesecond protein component, the efficacy of the rodent control agent maybe enhanced (relative to a rodent control agent comprising a singleprotein component conferring the rodent control activity). For example afusion protein, wherein the second protein component is acell-disruptive toxin (e.g. is selected from the group consisting ofperfringolysin O, alpha haemolysin, sphingomyelinase, delta-haemolysin,alpha toxin, cyt toxin, granulysin, melittin, perforin, equinatoxin,listeriolysin, aerolysin, haemolysin A, amoebapore, El Tor haemolysin,Vibrio damsela haemolysin, pneumolysin, streptolysin O, Kanagawa toxin,leptospira haemolysin, cry toxin, VIP3, thionin and β-purothionin) andthe further protein component(s) may be a toxin that requiresinternalisation within a cell for activity (e.g. selected from the groupconsisting of granzyme B, diphtheria toxin, cholera endotoxin, VIP2,accessory enterotoxin, colicin E1, CTX IV, a type 2 ribosomeinactivating protein such as ricin, anthrax toxin, Pseudomonas exotoxinA, barnase, a type 1 ribosome inactivating protein such as gelonin), maybe a particularly effective rodent control agent as themembrane-disrupting activity conferred by the second protein componentmay assist the functional activity of the further protein component byfacilitating access of the further protein component to the inside of arodent cell. In preferred embodiments the fusion protein will compriseat least one toxin component selected from the group consisting of:cyt2A, β-purothionin, and granulysin; and at least one toxin componentselected from the group consisting of granzyme B, VIP2A, and gelonin.

Fusion proteins of the invention may be constructed so that the firstprotein component (the antibody component) is N-terminal to the secondprotein component, alternatively they may be constructed so that thesecond protein component is N-terminal to the first protein component.As mentioned previously, in some embodiments it is desirable toindirectly link the two protein components via a linker peptide. Thusfusion proteins of the invention may comprise from N to C terminus, thefirst protein component linked via a peptide bond to a linker peptide,which is in turn linked via a peptide bond to the second proteincomponent, or they may comprise from the N to C terminus the secondprotein component linked via a peptide bond to a linker peptide, whichis in turn linked via a peptide bond to the first protein component.

Where the fusion protein comprises an additional protein component thismay be N-terminal to the first component i.e. the additional proteincomponent is linked either directly through its C-terminal residue via apeptide bond to the N-terminal residue of the first protein component,or indirectly via a peptide linker (or a further protein component) tothe N-terminal residue of the first protein component. In a furtherembodiment the additional protein component is N-terminal to the secondprotein component i.e. the additional protein component is linked eitherdirectly through its C-terminal residue via a peptide bond to theN-terminal residue of the second protein component, or indirectly via apeptide linker (or a further protein component) to the N-terminalresidue of the second protein component. In still further embodimentsthe additional protein component may be i) C-terminal to the firstcomponent i.e. the additional protein component is linked eitherdirectly through its N-terminal residue via a peptide bond to theC-terminal residue of the first protein component, or indirectly via apeptide linker (or a further protein component) to the C-terminalresidue of the first protein component; or C-terminal to the secondprotein component i.e. the additional protein component is linked eitherdirectly through its N-terminal residue via a peptide bind to theC-terminal residue of the second protein component, or indirectly via apeptide linker (or a further protein component) to the C-terminalresidue of the second protein component.

Antibody components and/or fusion proteins of the invention may beproduced by expressing nucleic acids encoding them in any suitableprotein expression system. Thus in a further aspect, the inventionprovides nucleic acids encoding fusion proteins of the invention.Nucleic acids encoding fusion proteins of the invention may obtained bycloning a nucleic acid encoding the first protein component in-framewith a nucleic acid encoding the second protein component. Where thefirst and second protein components are indirectly linked via a peptidelinker, a nucleic acid encoding the linker will separate and be in-framewith the two protein components of the fusion protein.

Nucleic acids encoding antibody components may be isolated usingstandard molecular biological techniques from hybridoma cells expressingan antibody of the invention. Alternatively, nucleic acids encodingantibody components may be obtained, again using standard molecularbiological techniques, from phage display or other library clones thatencode an antibody or antigen-binding fragment of the invention.

Nucleic acids sequence encoding examples of the second protein component(the toxic or contraceptive component) are available in the art, see forexample the EMBL database references in Table 2.

Nucleic acids encoding linker peptides may be synthesised de novo, forexample from oligonucleotides encoding the linker peptide sequence.

In order for an antibody component or fusion protein to be expressed ina suitable protein expression system the nucleic acid encoding thefusion protein will be operably linked to a suitable promoter, andoptionally a suitable transcription terminator. In general, the promoterto which the nucleic acid encoding the fusion protein is operably linkedwill be any promoter capable of driving expression of the fusion proteinin the host cell into which the nucleic acid is to be introduced. Thus,if it is desired that the antibody component or fusion protein beexpressed in a bacterial cell, the promoter will be operable in thatbacterial cell. Similarly if it is desired that the antibody componentor fusion protein be expressed in a fungal expression system thepromoter will be operable in a fungal cell and the same logic prevailsif the construct is to be introduced into a mammalian cell cultureexpression system or a plant expression system. The use of aseed-specific promoter is particularly desirable when the antibodycomponent or fusion protein is to be expressed in plant cells and/orplants. Where nucleic acids of the invention are operably linked to asuitable transcriptional terminator region, this will be one thatmediates the termination of transcription in the host cell in which theantibody component or fusion protein of the invention is to beexpressed. Transcriptional terminator regions suitable for this purposeare described in the art.

Suitable expression systems for expression of antibody components and/orfusion proteins of the invention include microbial expression systemssuch as bacterial (e.g. E. coli, Bacillus expression systems) and fungalsystems e.g. yeasts (such as, for example, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia species, Hansenula species), and otherfungal expression systems (e.g. filamentous fungal expressions systemsderived from Aspergillus species, Trichoderma reesei, and Neurosporacrassa); mammalian expression systems e.g. CHO cells, and plantexpression systems. Preferred plants and plant cells for use in theexpression of fusion proteins of the invention include wheat, barley,maize, sorghum, oats, rice and millet. The skilled man will be familiarwith these expression systems, which are described fully in the art.

Novel rodent control agents as described herein may also be in the formof a protein conjugate. Thus, in a further aspect the invention providesa protein conjugate comprising an antibody component of the invention asdescribed herein, chemically conjugated to a toxic component orcontraceptive component. In one embodiment the toxic component orcontraceptive component will be a small chemical entity, whilst in afurther embodiment the toxic component or contraceptive component willbe a protein or peptide as described hereinbefore with respect to fusionproteins of the invention. Where the toxic component is a small chemicalentity, examples of suitable toxic compounds for use in this aspect ofthe invention include colchicine; doxorubicin; calicheamicin; moleculesof the non-steroidal anti-inflammatory drug (NSAID) class; cytochalasin;anticoagulants such as brodificoum, difenacoum, bromadiolone,flocoumafen, difethialone, hydroxycoumarins, indane-diones; calciferol;bromethalin; flupropadine; zinc phosphide; scilliroside; sodium(mono)fluoroacetate; fluoroacetamide; alphachloralose; thalliumsulphate.

Where the contraceptive component is a small chemical entity, suitablehormones and hormone-like compounds for use in this aspect of theinvention include for example progesterones and oestrogens (bothsynthetic and natural) and diazacon (i.e. 20,25 diazacholesterol).

The antibody component of the protein conjugate may be obtained asdescribed previously and may be directly chemically conjugated to atoxic component or contraceptive component. Typically this conjugationwill involve the use heterobifunctional agents which result indisulphide or thioether linkages as described in the art (see forexample, Hermanson, G. T. “Bioconjugate Techniques” Academic Press,London, 1996, for standard methodologies relating to the use ofcross-linking reagents).

In a further embodiment where the toxic or contraceptive component is asmall chemical entity the toxic compound, hormone or hormone-likecompound may be encapsulated, and the capsule will be linked to anantibody or antigen-binding fragment of the invention. In one particularembodiment the capsule may be linked via chemical conjugation asdiscussed above. In another particular embodiment, the antibody orantigen-binding fragment of the invention may be chemically conjugated,or fused via a peptide bond, to a second binding component that binds tothe capsule. For example, an antigen-binding fragment of the inventionmay be used to provide one specificity in a bi-specific or evenmulti-specific binding molecule, wherein the second (or a further)specificity is for the capsule and this second (or further specificity)is provided by a molecule (e.g. an antigen-binding fragment) whichspecifically binds to the capsule.

In a further aspect the antibody component of the protein conjugate ischemically conjugated to two or more toxic or contraceptive components.The further toxic or contraceptive component may be in the form of aprotein or peptide moiety or in the form of a small chemical entity.Where at least one further toxic or contraceptive component is a proteinor peptide moiety, the further component may be a toxin, immunogen,hormone or proteinaceous hormone mimetic as described previously. Whereat least one further toxic or contraceptive component is a smallchemical entity it may be a toxic compound or a hormone or hormone-likecompound as described hereinbefore.

In certain embodiments of this aspect of the invention, proteinconjugates are created comprising an antibody component as describedherein, chemically conjugated to at least two further components, eachof which confers a rodent control (i.e. toxic or contraceptive, or both)function on the protein conjugate. The second further component (andfurther component or components) may (each) have the same, or differentrodent control functions to the first further component, and where ithas (they have) the same function it (they) may have the same, ordifferent (independently different), mode of action, as described abovewith respect to fusion proteins of the invention. In one particularembodiment the protein conjugate will comprise an antibody component asdescribed hereinbefore, chemically conjugated to one or more moleculesof a second toxic or contraceptive component. The site(s) of conjugationwill depend on the chemistry used for the conjugation reaction. Where itis desired that two different further components are conjugated to thefirst (antibody) component, it may be desirable to use a differentchemical method of conjugation for each further component in order toensure that the different further components do not compete with eachother for conjugation to the same site on the first component.

In yet a further aspect of the invention there is provided a rodentcontrol agent comprising a fusion protein or protein conjugate asdescribed hereinbefore, wherein the fusion protein or protein conjugatecomprises at least two antibody components. In one embodiment eachantibody component binds to the same target protein, thus increasing theprobability of the rodent control agent binding to its target tissue andalso potentially increasing the avidity of binding of the rodent controlagent. In such embodiments the antibody components may be identical ordifferent. Where the antibody components are different, this encompassesdifferent antibody components which bind to the same RSE or RSPE in atarget protein, or more preferably different antibody components witheach antibody component binding to a different RSE or RSPE within thesame target protein. In a further embodiment the rodent control agentwill comprise antibody components that bind to at least two differenttarget proteins. Embodiments comprising antibody components that bind todifferent RSEs or RSPEs within a single target protein and/or whichcomprise antibody components binding to different target proteins, maybe particularly useful in delaying the onset of resistance occurring tothe rodent control agent or in counteracting resistance at one of thepotential target sites.

Antibodies, antigen-binding fragments, fusion proteins and proteinconjugates of the invention are useful in controlling rodents, forexample they may be used in methods of killing rodents or in methods ofpreventing breeding in rodents. Thus in a further aspect there isprovided a rodent control agent comprising or consisting of an antibody,antigen-binding fragment, fusion protein or protein conjugate asdescribed herein.

Fusion proteins and protein conjugates of the invention, wherein thesecond protein component is a toxin or wherein the antibody orantigen-binding fragment is conjugated to a toxic compound orprotein/peptide toxin, will achieve rodent control by killing rodents(i.e. such fusion proteins and protein conjugates are rodenticidal intheir mode of action). Where the antibody or antigen-binding fragmentcomponent recognises a protein expressed in the epithelium of the GItract of rodents, the fusion protein or protein conjugate will bind tothe epithelium. Depending upon the type of toxin or toxic compoundpresent in the fusion protein/protein conjugate, the toxin or toxiccompound may disrupt the cell membrane. The integrity of the epitheliumof the GI tract is thus compromised, and lesions occurring in the GItract will lead to rodent death. Alternatively, where the toxin or toxiccompound mediates toxicosis through an intracellular mode of action, thebound fusion-protein or protein conjugate relies on internalisationthrough endocytosis. Once the fusion protein or protein conjugate hasentered the cell, the toxin/toxic compound will be able to mediatetoxicosis, which will ultimately result in death of the rodent.

Fusion proteins wherein the second protein component is an immunogenalso require uptake into rodent cells. Once present inside the rodentcell, an immune response is mounted against the immunogen. Thus wherethe immunogen is an ovum or sperm specific antigen, this results in thegeneration of an immune response that prevents reproduction fromoccurring. Advantageously the antibody or antigen-binding fragment ofthe fusion protein will increase the amount of immunogen that isabsorbed into the rodent (through endocytosis) and may also act as anadjuvant, thus increasing the likelihood of suitable immune responsebeing mounted.

Fusion proteins and protein conjugates of the invention wherein thesecond protein or conjugated component is a hormone or hormone-likecomponent similarly require uptake into rodent cells. Once presentinside a rodent cell, the hormone/hormone-like compound will interferewith the hormonal control of reproduction and will thus control rodentsby preventing breeding.

The rodent specificity of the antibody/antigen-binding part of thefusion proteins and protein conjugates described herein, confers severaladvantages on rodent control agents of the invention. The highspecificity for rodent tissue means that the rodent control agent isspecifically targeted to a rodent tissue thus facilitatinguptake/activity of the toxic/immunogenic/hormonal component. In turnthis specific targeting means that less rodent control agent is likelyto be required for effective control, less rodent control agent ispresent in the environment, and that which is present in the environmentis not specific for, and is thus less likely to be absorbed by and causedamage to, non-target species.

Rodent control agents of the invention may be formulated as acomposition comprising as the active ingredient a fusion protein orprotein conjugate of the invention in combination with at least oneadditive, diluent and/or carrier. Suitable additives include forexample, compounds that act as attractants to rodents, compounds whichmake the composition more palatable to rodents, additional rodentcontrol agents, and compounds which serve to stabilise or protect therodent control agent or agents. Suitable attractant compounds includefood materials such as wheat, barley, maize, sorghum, oats, rice andmillet. Suitable palatability enhancing compounds for use in theinvention include sweeteners, (e.g. acesulfame-K, alitame, aspartame,brazzein, cyclamate, saccharin, sucralose, sucrose, glucose, sorbitol,mannitol, xylitol, thaumatin, monellin, isomalt, and isomaltulose),vegetable or animal oils (e.g. maize, soybean and peanut oil, fish oil)and dried yeast. Suitable stability enhancing/protective compoundsinclude those which protect or prevent the rodent control agent oragents from proteolysis or hydrolysis upon ingestion by the rodent, forexample anatacids and compounds which may be used in the formation of pHrelease capsules.

Suitable diluents and carriers for use in compositions of the inventioninclude those that are used as diluents and/or carriers with knownrodent control agents, for example waxes, and binding agents e.g.cellulose ethers, starch, polyvinyl alcohol, polyvinyl pyrrolidone, guargum, carrageenan, gelatin, karaya gum; xanthum gum, acacia gum, locustbean gum, tragacanth, pectin and polyacrylates.

Compositions of the invention may comprise in addition to and/or insteadof any one of the aforementioned additives, diluents or carriers, anadditional rodent control agents such as those mentioned hereinbefore inthe introduction to the invention. In particular the present inventionalso includes mixtures of one or more of the novel rodent control agentsdescribed herein in combination with at least one first generationanticoagulant and/or at least one second generation anticoagulant.Preferred first generation anticoagulants for use in this aspect of theinvention include the hydroxycoumarin and the indane-diones, withwarfarin, coumachor, coumafuryl and coumatetralyl being particularlypreferred hydroxycoumarin and pindone, diphacinone, and chlorphacinonebeing particularly preferred indane-diones. Preferred second generationanti-coagulants for use in this aspect of the invention includebromadiolone, brodifacoum, difenacoum, flocoumafen, and difethialone.

The present invention also encompasses mixtures of at least two of thenovel rodent control agents as described hereinbefore, as well ascompositions (as described above) comprising such mixtures. For example,where a rodent control agent is an antibody component that binds to anextracellular epitope of a protein that is expressed in rodents (i.e.the antibody component per se is the functional rodent control agent),this may be combined with one or more further rodent control agents,wherein the further rodent control agent comprises an antibody componentand one or more toxic or contraceptive components (i.e one or morefurther rodent control agent(s) is(are) a fusion protein or proteinconjugate as described herein). In further embodiments the mixture willcomprise two or more fusion proteins and/or protein conjugates asdescribed herein.

In one embodiment fusion proteins of the invention are produced inplants and the plant material containing the expressed fusion protein isused as bait for rodent control. Preferably the plant producing thefusion protein will be one that is capable of acting as a source of foodfor rodents, for example, wheat, barley, maize, sorghum, oats, rice andmillet are all suitable plants for the expression of fusion proteins ofthe invention, according to this aspect of the invention. In oneembodiment it is particularly preferred that the fusion protein will beexpressed in the seeds of the plant. In this way the grain from plantsexpressing a fusion protein of the invention may be used directly asbait.

As mentioned hereinbefore compositions of the invention, as well asplants and/or grain containing fusion proteins of the invention, may beused as rodent control agents. Accordingly in yet a further aspect, theinvention provides a method of killing rodents, comprising placing arodent control agent in an area frequented by a rodent, such that uponingestion by said rodent of said rodent control agent, said rodent iskilled. The skilled man will also appreciate the invention also providesa method of preventing rodents from breeding comprising placing a rodentcontrol agent in an area frequented by a rodent, such that uponingestion by said rodent of said rodent control agent, the reproductivecapability of said rodent is inhibited.

In order to test the efficacy of the rodent control agents of theinvention various in vitro and in vivo studies may be conducted. Anexample of a suitable in vitro test is the gut loop assay as describedby Heylings 1991 (Toxicol. Appl. Pharmacol. 107:482-293) and in Example9.

Typical strategies for the in vivo evaluation of the rodenticidalproperties of rodent control agents of the invention are based on thefollowing requirements: 1) to minimise the number of animals used intests, 2) to keep costs down, 3) to produce useful information quickly,4) not to reject active substances that may have promise.

Initial testing will usually be conducted in the laboratory because testconditions can be carefully controlled. A cascade of test procedures isused. This cascade allows active substances to be accepted or rejectedby using a sequential decision process. The usual test subjects are theNorway rat, Rattus norvegicus, and the House mouse, Mus domesticus. TheRoof rat is another important test subject but as it is not available asa laboratory strain it is tested only in the latter stages of anevaluation programme. Strains of rodents that are resistant toanticoagulants may also be used in the later stages of a laboratoryprogramme in order to test efficacy against these animals.

Rodent control agents of the invention that are active and show promisein laboratory tests may then be evaluated in the field. Field trials areconducted against all important pest rodent species in a variety ofnatural circumstances.

The skilled man is referred to the guideline documents that are readilyavailable and which set out logical testing procedures for rodenticidesin the laboratory (EPPO/OEPP. 1999. Laboratory tests for evaluation ofthe toxicity and acceptability of rodenticides and rodenticidepreparations; OEPP/EPPO PP 1/113(2): 89-101) and field (EPPO/OEPP. 1999.Guidelines for the Efficacy Evaluation of Plant Protection Products:Field-tests against synanthropic rodents Mus musculus, Rattusnorvegicus, R. rattus; OEPP/EPPO PP 1/114(2):102-113). Recommendationsare also available for test procedures that satisfy regulatoryrequirements in the UK (Anonymous. 2005. Guidelines on the Efficacy DataRequirements for Approval of Non-agricultural Pesticide ProductsRodenticides. Health and Safety Executive, Bootle, UK. 30 pp), theEuropean Union (Anonymous. 2002. Technical notes for guidance in supportof the Annex VI of Directive 98/8/EC of the European Parliament and theCouncil concerning the placing of biocidal products on the market.Common principles and practical procedures for the authorisation andregistration of products. European Commission, July 2002. 215 pp.) andthe United States.

A typical cascade of tests which may be followed in the laboratory inorder to assess the efficacy of the rodent control agents of theinvention may include oral intubation tests, no-choice feeding tests,and choice feeding tests. Further details of these tests are outlinedbelow.

Oral Intubation: initial tests to establish the potency of activesubstances involve the delivery of the active substance, carried in aninert liquid such as polyethylene glycol, directly to the stomach oftest subjects using a gavage. Exact doses can be delivered in this wayin order to determine lethal dosage percentile statistics. The testprovides information on the capability of the active substance to remainactive in the conditions found in the gut of the subject species and onits transfer across gut membranes. The test also provides information onthe intrinsic toxicity of the active substance.

No-choice Feeding Tests: virtually all commercial rodenticide productsare presented as formulated baits. The next stage of the test cascadeinvolves the preparation of a bait containing the active substance (inthis case a rodent control agent of the invention). A ‘no-choice’ test,in which individually-caged test subjects have only the experimentalbait presented to them, is first conducted to establish whether theactive substance is active when delivered as an edible bait. The testbait is normally available ad libitum. In addition to information likethat obtained in oral intubation tests, no-choice feeding tests provideinformation on the capability of the active substance to be removed fromthe bait by the digestive processes of the subject species.

Choice Feeding Tests: to be capable of being controlled by an activesubstance in a formulated bait, rodents must consume sufficientquantities of the bait to acquire a lethal dose in the presence ofalternative natural foods. Therefore, the palatability of the activesubstance (i.e. a rodent control agent of the invention) is an importantaspect of evaluation. Choice tests are conducted in which the activesubstance is added to a bait base in measured concentrations.Individually-caged test subjects are then offered a choice between atest bait containing the active substance and an identical bait withoutthe active substance. A series of such tests is conducted to establishthe concentration of the active substance which is detected by the testsubjects. Normally, such detection is demonstrated by an aversion to thebait containing the active substance. An important consideration inevaluation is that the concentration detected by the test subjects, andwhich elicits significant aversion, is lower than the concentrationrequired to deliver a lethal dose in a test bait.

Further choice tests are conducted on experimental baits that areproduced during the development of commercial formulations. These choicetests involve the presentation of the experimental formulation and a‘challenge diet’. The challenge diet is composed so that it presents areasonably palatable alternative to the experimental formulation. Atypical ‘challenge diet’ used in such tests is ‘EPA meal’. This is aformulation comprising fixed quantities of oats, maize grits, sugar andoil. It is normal practice that an experimental formulation whoseconsumption comprises a minimum of 30% of the combined challenge dietand experimental formulation consumed by the test subjects would beconsidered a potential candidate for field trials.

Following laboratory testing, efficacy may be tested in a fieldenvironment. Rodents have complex and highly adaptive behaviours whichare only fully exhibited under natural conditions. Therefore, fieldtrials may be conducted in order to assess the effectiveness ofrodenticide active substances and formulated products under fieldconditions. Normally, field trials are carried out against a range oftarget species in a variety of natural environments that are typical ofthose in which practical rodent control treatments might be conducted.

Field trials may be conducted at an early stage of product developmentusing experimental formulations, not intended for commercialisation, inorder to investigate natural behavioural processes in the target rodentspecies when they are presented with a typical bait containing theactive substance.

Field trials may also be carried out subsequently on commercial baitproducts to demonstrate their efficacy under practical conditions.

Various aspects and embodiments of the present invention will now beillustrated in more detail by way of example. It will be appreciatedthat modification of detail may be made without departing from the scopeof the invention.

For the avoidance of doubt, literary reference, patent application, orpatent, is cited within the text of this application, the entire text ofsaid citation is herein incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Inhibition of luciferase translation by recombinant gelonin.

FIG. 2 Inhibition of luciferase translation by anti-CNT2 polyclonalantibody-recombinant gelonin conjugate. Pool A samples correspond to thepooled fraction from the IMAC purification which were determined tocontain free antibody. Pool B samples correspond to the pooled fractionsfrom the IMAC purification which were determined to containantibody-toxin conjugate.

EXAMPLES Example 1 Generation and Preparation of RSPES 1.1 PeptideSelection and Synthesis

Potential protein targets, present on the gut epithelium of rodents areidentified by literature and bioinformatic approaches. Rodent specificpeptide epitopes (RSPEs) are identified in these proteins based on thefollowing criteria: high sequence identity (preferably between 80 and100% identity) between mouse and rat sequences and low sequence identity(preferably between 0 and 40% identity) between rodent and humansequence, and no significant hits with other species using BLASTalignments; their hydrophilicity profiles (indicator of surfaceprobability); predictions of flexibility and secondary structure.Algorithms used for predicting hydrophilicity, flexibility and secondarystructure are provided by programmes in the DNAstar and Vector NTIsuites of programmes.

Once a suitable RSPE has been identified, the peptide is synthesisedusing Fmoc solid phase synthesis and purified to around 90% purity. Thepeptides shown in Table 3 below have been synthesised.

Where suitable, an N-terminal cysteine is added to the sequence toenable directed conjugation to a carrier protein. Alternatively, andwhere suitable, the sequence is synthesised with the N-terminal aminoacid left unblocked to enable directed conjugation to a carrier protein.When not required for conjugation, the N-terminal amino acid is blockedby acetylation. In addition the C-terminal amino acid is blocked with anamide group.

TABLE 3 RSPEs that have been synthesised RSPE primary amino SourceProtein acid sequence SEQ ID NO: Rat oilgopeptide VIRSRASDGCLEVKE SEQ IDNO: 1 transporter PepT1 Rat oligopeptide CSSDFKSSNLD SEQ ID NO: 2transporter PepT1 Rat CD155 (PVR, SNVNGSYREMKETGSQP SEQ ID NO: 3 Tage4)Rat GTR2 (GLUT2) GTDTPLIVTPAHTTP SEQ ID NO: 4 glucose transporter RatCFTR chloride LKNNPVNGGNNGTKIA SEQ ID NO: 5 transporter Rat CNT2 nucleo-WQDKESSLRNLAK SEQ ID NO: 6 side transporter Rat CATB(O+) GGDMFMNISWVNSEQ ID NO: 7 (slc6a14) colonic amino acid transporter Rat MDR1 multidrugSFTPSRDPHSDRAIT SEQ ID NO: 8 resistance transporter Mouse MDR1 multi-SFTKAEASILPSIT SEQ ID NO: 9 drug resistance transporter Rat Sucrase-YNAESITNENAGLKATL SEQ ID NO: 10 Isomaltase Mouse GLUT7 NTPHKVLKSFYN SEQID NO: 11 glucose transporter Mouse GLUT7/Rat YYDRNKENIES SEQ ID NO: 12GTR5 (GLUT5) glucose transporters Rat Npt2a PETKEASTSMSRVEA SEQ ID NO:13 (Slc34a1) sodium/ phosphate transporter IRat OATP-B QPGPSLFPGCSEPCSCQSEQ ID NO: 15 (SLC21A9) organic anion transporting polypeptide Rat DRA1chloride/ LSSSSAENDSMIEEKVMV SEQ ID NO: 24 anion exchanger Rat ENT1equili- TNQSCESTEALADPSVSL SEQ ID NO: 26 brative nucleoside transporterRat GCC (Guanylyl VSGRFPSERS SEQ ID NO: 27 cyclase) Rat PLBAEDLWIQAKELVRHLKDNP SEQ ID NO: 28 (Phospholipase B) Rat LPH (lactase-EDAAPTASPVQS SEQ ID NO: 29 phlorizin hydrolase) Rat AMPN GSTSATTSTTNPASEQ ID NO: 31 (aminopeptidase N) Rat MCDL (mucin NKDILLTTVPMETERTIR SEQID NO: 32 and cadherin-like protein) Rat SCAB SSNPAPGST SEQ ID NO: 34(amiloride- sensitive sodium channel beta- subunit)

1.2 Conjugation of Peptides to BSA

Peptides containing a N-terminal cysteine residue are coupled to BSAusing the hetero-bifunctional crosslinking agentm-Maleimidobenzoyl-N-hydroxylsuccinimide ester. Coupling is via theprimary amines on lysine residues in BSA and sulphydryl groups oncysteine residues in the peptide as described by Kitagowa & Aikawa. J.Biochemistry Vol 79: pp 233-236 (1976).

A 25 mg/ml solution of BSA (Sigma, Poole, Dorset) is prepared in 0.2Msodium phosphate, pH 7.0 and a 25 mg/ml solution of MBS (Perbio,Cheshire) made up in dimethyl formamide (Sigma). 30 μl of MBS solutionis added drop wise to 1 ml of the BSA solution with mixing and incubatedin the dark at room temperature for 45 minutes. The activated BSAsolution is then passed down a PD10 gel filtration column (GEHealthcare, Buckinghamshire), which had been previously equilibratedwith 0.2M sodium phosphate, pH 7.0. The BSA containing fractions areidentified by absorbance at 280 nm and pooled. 2 mg of peptide isdissolved in 1 ml of 50 mM sodium phosphate, pH 7.5. Sufficientactivated BSA solution is added to the peptide to achieve a 30:1 molarratio of peptide:BSA. This is incubated at room temperature for 4 hours,then overnight at 4° C. in the dark with mixing. Conjugated peptides arestored at −20° C.

Peptides that contain lysine residues or primary amines at theN-terminus are coupled to BSA using the 2-step glutaraldehyde method.Coupling is between the primary amine groups in the BSA and peptidebased on the method in Bio-conjugate Techniques, Academic Press 1996, pp583-584.

A 10 mg/ml solution of BSA is made up in 0.1M sodium phosphate, 0.15Msodium chloride pH 6.8. Glutaraldehyde (Sigma) is added to a finalconcentration of 1.25% and the mixture incubated with mixing for 12hours at room temperature. The activated BSA is passed down a PD10 gelfiltration column, which had been previously equilibrated with PBS. TheBSA containing fractions are identified by absorbance at 280 nm andpooled. 2 mg of peptide is dissolved in 1 ml of 0.5M sodium carbonate pH9.5 and sufficient activated BSA solution is added to the peptide toachieve at least a 10:1 molar ratio of peptide:BSA. The mixture isincubated overnight at 4° C. Excess reactive sites are blocked byaddition of 40 μl of 1M ethanolamine (Sigma). Conjugated peptides arestored at −20° C.

Example 2 Generation of Antibody Components

Peptides that have been synthesised and conjugated to BSA as describedin Example 1 above are used to generate antibodies that bind to anextracellular epitope of a protein expressed in a rodent.

2.1 Rabbit Immunisation Protocol

New Zealand white rabbits are used for polyclonal antibody production.Rabbits are immunised with 100 μg of protein administered subcutaneouslyusing Freunds Complete adjuvant (Sigma) for the first dose. This isfollowed by three booster immunisations on days 28, 56 and 84 containing100 μg of protein administered subcutaneously in Freunds Incompleteadjuvant (Sigma). Pre-bleeds are taken prior to the initialimmunisation, test bleeds are taken 10-14 days post the third dose andharvest bleeds are taken 10-14 days post the fourth dose.

For each of the following RSPEs two rabbits were immunised andpolyclonal antibody sera generated: SEQ ID NO:1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 15.

For each of the following RSPEs one rabbit was immunised: SEQ ID NO: 27,SEQ ID NO: 28; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:34.

2.2 Mouse/Hamster Immunisation Protocol

Mice and hamsters are used for monoclonal antibody production, and areimmunised with 20 μg of protein administered subcutaneously in FreundsComplete adjuvant for the first dose. This is followed by twoimmunisations of 20 μg of protein administered subcutaneously on days 28and 56. Doses are administered in Freunds Incomplete adjuvant at day 28and in phosphate buffered saline (PBS) at day 56. Test bleeds taken 7days post the dose 3. At least 6 weeks after the third dose mice areboosted with 20 μg of protein administered intravenously in PBS. Spleensare harvested for fusions 4 days later.

Mice have been immunised with each of the following RSPEs and polyclonalantibody sera generated: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29.

2.3 Production of Monoclonal Antibodies

Monoclonal antibodies are generated using a method based on Kohler G. &Milstein C. Nature 256, 495-497 (1975). Lymphocytes are washed from theharvested spleens with Dulbecco's Modified Eagle's Medium (DMEM,Invitrogen, Paisley) delivered by syringes with 20-gauge needles. NS0myeloma cells (European Collection of Cell Cultures, Porton Down,Salisbury) are cultured in DMEM containing 10% (v/v) foetal bovine serum(PAA, Yeovil, Somerset), 2 mM L-glutamine, 1×HT supplement and 50units/ml penicillin and 50 ug/ml streptomycin (all from Invitrogen) at37° C. in 5% CO₂ to a density of 5×10⁵/ml. Lymphocytes from a singlespleen (approximately 2×10⁸) will be mixed with 2×10⁷ NS0 myeloma cells.Following centrifugation at 2000×g for 4 minutes, the cell pellet isgently resuspended and fused by dropwise addition of 1 ml of a 50% (w/v)polyethylene glycol (1500) in 75 mM HEPES buffer pH 8 (Roche, Lewes,East Sussex) over one minute. Complete culture medium (as describedabove) supplemented with H1 cloning supplement (Roche) is added slowlyover several minutes to a final volume of 50 ml. The resulting fusionsolution is cultured in five sterile microtitre cell culture plates(Nunclon, Fisher, Loughborough, Leicestershire) at 100 ul/well for 4hours at 37° C. in 5% CO₂ after which, 100 ul/well of complete culturemedium containing 4% (v/v) 1×HAT selection medium (Invitrogen) is added.Fourteen days post fusion the culture supernatants are assayed forpeptide specific antibodies using an antibody capture enzyme-linkedimmunosorbant assay (ELISA) based on the methods described by EngvallE., and Perlmann P. Immunochemistry 8, 871-874 (1971); Harlow E. et al.,Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988 pp.182-183. Selected hybridoma cells are taken through at least two roundsof cloning by limiting dilution followed by re-assay, to ensure bothclonality and stability of the hybridomas. Banks of frozen hybridomasare prepared in a freezing medium composed of 10% (v/v) DMSO (Sigma,Poole, Dorset) in foetal bovine serum at a freezing rate of 1° C./minutefor 80 minutes followed by storage in liquid nitrogen. Production ofselected monoclonal antibodies is achieved by scaling-up tissue culture.

Hybridoma fusions have been obtained using lymphocytes from the spleensof mice immunised with SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 11, SEQ IDNO 6 and SEQ ID NO 27. 4 Stable monoclonal cell lines producingantibodies against the RSPE from mouse GLUT7 (SEQ ID NO 11) have beenisolated, grown and frozen. 4 Stable monoclonal cell lines producingantibodies against the RSPE from Rat CFTR (SEQ ID NO 5) have beenisolated. Seven positive monoclonal cell lines producing antibodiesagainst an RSPE from Rat PepT1 (SEQ ID NO 1) have been isolated, grownand frozen.

2.4 Identification and Isolation of Antibody Components fromPhage-Display Libraries2.4.1 Screening of Naïve Camelid V_(HH) Phage Display Library

RSPEs are prepared for screening of a naive Llama glama V_(HH) M13filamentous phage-display repertoire by chemical synthesis andsubsequent conjugation to BSA, as described in Example 1.2 above.Screening of a phage-displayed immune binding-domain repertoire followsestablished procedures, such as those described by McCafferty & Johnson(in “Phage Display of Peptides and Proteins”, pg 98-100, Eds. Kay,Winter & McCafferty, 1996, Academic Press, Inc.). RSPE::BSA conjugatesare adhered to the interior surface of Maxisorb Immunotubes (Nunc) byapplication in 50 mM sodium hydrogen carbonate pH 9.6 buffer at aconcentration of 100 μg/ml and overnight incubation. Free RSPE::BSAconjugates are removed by three rinses in PBS, and then blocking of thesurface by addition of PBS/2% skimmed milk protein (PBSM) and incubationat 37° C. for two hours. Following sensitisation of immunotubes with therequired RSPE “panning” is effected by the addition of 10¹² to 10¹³phage in 4 ml of PBSM and incubating for two hours at room temperature.After this step, tube contents are aspirated and the tube is washedtwenty times using PBS/0.1% Tween 20, followed by a further twentywashes with PBS. Immunotube-bound phage (representing a majority ofRSPE::BSA-specific V_(HH) binders) are then eluted by the addition of 1ml 100 mM glycine pH 3.0 for 10 minutes. The solution is transferred toa fresh tube containing 0.5 ml 1M Tris-HCl pH 7.4 to neutralise. Theeluted phage are then used to infect log-phase TG1 E. coli cells bymixing and incubating at 37° C. for 30 minutes, followed by spreading ona 24×24 cm 2×TY/2% glucose/100 μg/ml ampicillin plate (Nunc BioAssayDish) which is then incubated at 37° C. overnight. The next day theplate is scraped to remove cells (which represent an enriched,increased, population of phagemid clones) containing sequences withbinding specificity for the RSPE::BSA conjugate. In order to furtherscreen/select clones from this population which demonstrate highaffinity for the RSPE, phage particles are “rescued” by the addition ofhelper phage, e.g. M13-KO7, or VCS-M13. Briefly, cells from the laststep are inoculated into 50 ml of 2×YT/2% glucose/100 μg/ml ampicillinin a 250 ml conical flask and incubated at 30° C. until the OD₆₀₀ isabout 0.5. Helper phage are then added to provide a 1:1 ratio of helperphage to bacteria, typically 2×10¹⁰ pfu/50 ml, followed by incubation at37° C. for one hour. Next, cells are sedimented by centrifugation at3000 g for 10 minutes, the supernatant is discarded, the cell pellet isresuspended and used to inoculate fresh medium in a 2 litre flask: 500ml 2×YT/100 μg/ml ampicillin/50 μg/ml kanamycin (no glucose). Thisculture is incubated overnight at 30° C. with vigorous shaking andphage-V_(HH) particles are then harvested by the addition of 100 ml 20%PEG/2.5M NaCl for 30 minutes at 4° C. Precipitated phage are thencollected by centrifugation at 4000 g for 10 minutes and resuspended in5 ml PBS, ready for the next round of screening. The efficiency of theselection process may be improved by the reduction of the amount ofRSPE::BSA conjugate used to sensitise the immunotube at each round ofpanning. In addition, the selection process can be adjusted to includesteps which will bias the selection of phage-V_(HH) particles thatexhibit certain physico-chemical characteristics, such as proteolyticstability. For example, it is known that M13 phage particles areresistant to proteolysis by certain proteolytic enzymes, such as trypsinand chymotrypsin (Schwind et al, 1992, Eur. J. Biochem. 210: 431-436),and that this property can be exploited in phage-display for theselection of structurally stable variants (Kristensen & Winter, 1998,Folding & Design, 3:321-328).

In order to select individual clones which exhibit the desired affinityand selectivity for the RSPE, unique colonies on a 2×TY/2% glucose/100μg/ml ampicillin plate (obtained from serially-diluted samples of theTG1 E. coli infected by eluted phage after a panning step) are pickedand arrayed in a 96 well culture block containing 150 μl 2×TY/2%glucose/100 μg/ml ampicillin per well, and incubated with shaking at 30°C. until OD₆₀₀ reaches about 0.5. Helper phage are then added to eachwell to provide a 1:1 ratio of helper phage:bacteria, followed byfurther incubation with shaking at 37° C. for one hour. Then the mediumis removed after sedimenting cells by centrifugation at 3000 g, andreplaced with 1.5 ml of 2×YT/100 μg/ml ampicillin/50 μg/ml kanamycin (noglucose) and incubation is allowed to continue with vigorous shaking at30° C. overnight. Phage particles are then harvested from each wellusing 20% PEG/2.5M NaCl for 30 minutes at 4° C. followed bycentrifugation at 4000 g and resuspension in PBS. These clonal phagesamples are either used in an ELISA to determine the relative bindingaffinity for immobilised RSPE, using an anti-phage antibody-HRP complex,or, alternatively, soluble V_(HH) are expressed by infection of anon-amber suppressor E. coli host, (such as strain HB2151) with thephage. 96 well culture and expression of soluble, secreted V_(HH) isthen made possible using IPTG induction, and either crude culture medium(containing soluble V_(HH)), or, IMAC-purified V_(HH) (by virtue of anintegral hexa-histidine tag) is used in ELISA to determine binding toimmobilised RSPE. A suitable detection antibody in this case is 9E10-HRP(Roche Molecular Biochemicals) which detects the c-Myc tag present onthe soluble V_(HH) protein.

Initially the following RSPEs are screened as RSPE::BSA conjugatesagainst a naïve Camelid phage display library as described above: RSPEwith SEQ ID NO 5, RSPE with SEQ ID NO: 6, RSPE with SEQ ID NO: 11 andRSPE with SEQ ID NO 27. The V_(HH) clones identified from this screenare then used in the production of fusion proteins and antibodyconjugates as described herein.

2.5 Cloning and Expression of Recombinant Antibody Binding Domains

To exemplify the process of creating single chain antibodies, morespecifically scFv, the SV63 mouse monoclonal antibody (MAb) (recognisinga cell-surface epitope from human alkaline phosphatase expressed bycertain colorectal tumours) from the hybridoma HB-8766 (American TypeCulture Collection; Rettig et al, 1989, U.S. Pat. No. 4,851,332) is usedas a model cell-surface antigen binding protein. In order to derivatisean scFv molecule from this IgG1 MAb the Fv sequences are cloned from thehybridoma using RT-PCR with rodent Fv and constant domain-specificprimer sets as described in the literature (e.g. Dubel et al. 1994,Journal of Immunological Methods 175: 89-95; McCafferty & Johnson in“Phage Display of Peptides and Proteins”, pg 95, Eds. Kay, Winter &McCafferty, 1996, Academic Press, Inc.), using the modifications toindividual primers as specified in the literature. The sequences of theprimers used in this Example are given in Table 4 below.

TABLE 4 Primers used in cloning the SV63 scFvs from hybridomas ATCCHB-8766 Primer Sequence 5′ to 3′ SEQ ID RoPro-9 AGGTSCAGCTGCAGSAGTCWGGSEQ ID NO: 37 RoPro-28 CCAGGGGCCAGTGGATAGACAGATGGGGGTGTCGTT SEQ ID NO:38 TT RoPro-6 GAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTG SEQ ID NO: 39RoPro-25 TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC SEQ ID NO: 40 RoPro-3GGTGATATCGTKCTCACYCARTCTCCAGCAAT SEQ ID NO: 41 RoPro-4GGGAAGATGGATCCAGTTGGTGCAGCATCAGC SEQ ID NO: 42 scFv oligo 1AGCCCGCCATGGCCGATATCGTTCTCACTCAATC SEQ ID NO: 43 scFv oligo 2CGCCAGAGCCACCGCCACCGCTACCGCCACCGCCCT SEQ ID NO: 44 TGATCTCCAGTTTGGTGCCTCscFv oligo 3 AGCGGTGGCGGTGGCTCTGGCGGTGGCGGTAGCGA SEQ ID NO: 45GGTCCAGCTGCAGGAGTCTGG scFv oligo 4 CTATGAATTCAGTGGTGGTGGTGGTGGTGCTTGTCGTSEQ ID NO: 46 CGTCGTCCTTGTAGTCTGAGGAGACTGTGAGAGTGG TGC scFv oligo 5AGCCGGCCATGGCCGAGGTCCAGCTGCAGGAGTCTG SEQ ID NO: 47 scFv oligo 6CGCCAGAACCAGCTCCGCCGCTTCCGCCACCGCCTG SEQ ID NO: 48AGGAGACTGTGAGAGTGGTGCC scFv oligo 7 AGCGGCGGAGGTGGTTCTGGCGGTGGCGGAAGCGASEQ ID NO: 49 TATCGTTCTCACTCAATCTC soFv oligo 8GTATGAATTCAGTGGTGGTGGTGGTGGTGCTTGTCGT SEQ ID NO: 50CGTCGTCCTTGTAGTCCTTGATCTCCAGTTTGGTGCC TC2.5.1 Isolation of Hybridoma RNA and cDNA Synthesis

Hybridoma cells (10⁷) were centrifuged and resuspended in 1 ml ofTrizol™ and 200 μl of chloroform. The sample was mixed vigorously for 15minutes at room temperature and then centrifuged at 12000 g for 15minutes at 4° C. The aqueous layer was removed and an equal amount ofisopropanol is added. The sample was centrifuged at 12,000 rpm for 15minutes at 4° C. to precipitate the RNA, which was washed in 70% ethanoland then resuspended in RNase free water.

Traces of genomic DNA were removed from RNA by treatment with RNase freeDNase in a 10 μl reaction containing 5 μl RNA, 0.1 U RNase free DNase I(Ambion) and 1 U RNasin. The mixture was placed at 37° C. for 30 minutesfollowed by an incubation at 80° C. for 5 minutes.

The DNase I treated RNA was then used in an Accuscript (Stratagene)reaction under the manufacturer's standard conditions (1.5 μl RNA, 50 μltotal reaction volume, oligo-dT primer) to produce 1^(st) strand cDNA.

2.5.2 Isolation and Cloning of SV63 V_(H) Region

PCR was carried out using 1 μl of the oligo-dT primed 1^(st) strand cDNAderived from SV63 RNA as template and oligonucleotides RoPro-9 (SEQ IDNO: 37) and RoPro-28 (SEQ ID NO: 38).

The Roche GC-RICH PCR System was used with GC-RICH resolution buffer ata concentration of 0.5M in a reaction volume of 25 μl.

The reaction was carried out in a Stratagene Robocycler with thefollowing cycling conditions:

Cycle 1-5 Cycle 6-35 Final extension 94° C. 30 sec 94° C. 30 sec 54° C.30 sec 60° C. 30 sec 72° C. 60 sec 72° C. 60 sec 72° C. 300 sec

The yield of PCR product was very low from this reaction. Hence, asecond PCR was carried out to further amplify molecules produced.

Using the 2 μl of the PCR product from the reaction described above astemplate, two reactions were set up using the following oligonucleotideprimer pairs: i) RoPro-6 (SEQ ID NO 39, less specific than RoPro-9 (SEQID NO 37) and RoPro-25 (SEQ ID NO: 40, 3′ nested) and ii) RoPro-9 (SEQID NO: 37) and RoPro-28 (SEQ ID NO: 38). The PCR was carried out asdescribed above.

When samples of the above reactions were run on a 1% agarose/TBE gel andstained with ethidium bromide, both PCRs contained DNA fragments ofapproximately 400 bp. The PCR product resulting from reaction 2 abovewas isolated and purified using Geneclean Spin kit and cloned intopCRTOPO BluntII (Invitrogen).

Eight TOPO clones were fully characterised by DNA sequencing, one ofthese clones had the characteristics of a typical (but unique) V_(H)sequence as determined by BLASTP analysis of the UniProt/IPI-databases.

2.5.3 Isolation and Cloning of SV63 V_(L) Region

PCR was carried out using 20 of the oligo-dT primed 1^(st) strand cDNAderived from SV63 RNA as template and oligonucleotides RoPro-3 (SEQ IDNO: 41) and RoPro-4 (SEQ ID NO: 42). Stratagene Pfu Ultra DNA polymerasewas used in a reaction volume of 50 μl and the reaction was carried outin an MJ Research Dyad thermocycler using the following cyclingconditions: 95° C., 1 min then [95° C., 1 min; 52° C., 1 min; 68° C., 3mins] for 40 cycles, followed by a final extension of 68° C., 10 mins.

A sample of the above reaction was analysed on a 1% agarose/TBE gel andstained with ethidium bromide, a fragment of approximately 350 by wasobserved. This PCR product was isolated and purified using QiaQuick(Qiagen) gel elution and cloned into pCRTOPOBlunt II (Invitrogen).

Five TOPO clones were fully characterised by DNA sequencing, four ofthese clones were identical and had the characteristics of a typical(but unique) V_(L) sequence as determined by BLASTP analysis of theUniProt/IPI databases. One of the five clones contained a sequence thatwas identical to the MOPC21 kappa light chain variable sequence(Swissprot: P01634), an irrelevant sequence amplified from the myelomafusion partner used in the creation of the hybridoma.

2.5.4 Assembly of V_(H) & V_(L) Sequences into scFv Constructs

PCR-overlap extension (also known as “Splice-overlap extension”; SOE)was used to create two orientations of SV63 scFv: (i) V_(H)-[Gly₄Ser]₃linker-V_(L) and (ii) V_(L)-[Gly₄Ser]₃ linker-V_(H)), each containingN-terminal PelB leader sequences and C-terminal FLAG and hexa-histidinetags in the E. coli expression vectors pDGF (derived from the NEB vectorpMALc-2, pDGF contains all of the features of pMALc-2 except for thesequence encoding the maltose binding protein, which has been excised)and pIMS147 (Hayhurst & Harris, 1999, Protein Expression andPurification 15: 336-343). Table 5 below indicates the oligonucleotideprimers used and their purpose.

TABLE 5 Primers used in the assembly of V_(H) and V_(L) sequences intoscFv constructs Primer Purpose SEQ ID NO: scFv oligo 1 SV63 scFv VL::VHorientation PelB:VI forward primer 43 scFv oligo 2 SV63 scFv VL::VHorientation VI:GlySer reverse primer 44 scFv oligo 3 SV63 scFv VL::VHorientation GlySer:Vh forward primer 45 scFv oligo 4 SV63 scFv VL::VHorientation Vh:FLAG:His reverse primer 46 scFv oligo 5 SV63 scFv VH::VLorientation PelB:Vh forward primer 47 scFv oligo 6 SV63 scFv VH::VLorientation Vh:GlySer reverse primer 48 scFv oligo 7 SV63 scFv VH::VLorientation GlySer:VI forward primer 49 scFv oligo 8 SV63 scFv VH::VLorientation VI:FLAG:His reverse primer 50

To create the V_(L)-[Gly₄Ser]₃ linker-V_(H) scFv sequence, PCR wasperformed in two steps. The first step consisted of two reactions usingi) scFv oligo's 1 & 2 (SEQ ID NOs: 43 and 44, respectively) and apCRTOPOBluntII SV63 V_(L) clone as template to amplify the 5′ half ofthe construct; ii) scFv oligo's 3 & 4 (SEQ ID NOs: 45 and 46respectively) and a pCRTOPOBluntII SV63 V_(H) clone as template toamplify the 3′ half of the construct. The second step used SOE to jointhe two fragments from (i) and (ii) by annealing their complementary 3′and 5′ termini (respectively) and extending to full-length product bythe addition of polymerase, with subsequent amplification using scFvoligo's 1 & 4 (SEQ ID NOs: 43 and 46 respectively).

The V_(H)-[Gly₄Ser]₃ linker-V_(L) scFv sequence was created in a similarapproach. The first step consisted of two reactions using i) scFvoligo's 5 & 6 (SEQ ID NOs: 47 and 48, respectively) and a pCRTOPOBluntIISV63 V_(H) clone as template to amplify the 5′ half of the construct;ii) scFv oligo's 7 & 8 (SEQ ID NOs: 49 and 50, respectively) and apCRTOPOBluntII SV63 V_(L) clone as template to amplify the 3′ half ofthe construct. The second step used SOE to join the two fragments from(i) and (ii) by annealing their complementary 3′ and 5′ termini(respectively) and extending to full-length product by the addition ofpolymerase, with subsequent amplification using scFv oligo's 5 & 8 (SEQID NOs 47 and 50, respectively).

First step reactions were performed using a Roche GC RICH kit and themanufacturer's conditions with GC-RICH resolution buffer at aconcentration of 0.5M in a reaction volume of 25 μl. The reactions werecarried out in a Stratagene Robocycler with the following cyclingconditions:

Cycle 1-2 Cycle 3-27 Final extension 94° C. 30 sec 94° C. 30 sec 54° C.30 sec 65° C. 30 sec 72° C. 60 sec 72° C. 60 sec 72° C. 300 sec

Samples of the above reactions were analysed on a 1% agarose/TBE gel andstained with ethidium bromide and these revealed that all four expectedDNA fragments had been produced.

Second step SOE reactions were carried out by mixing the appropriatefragment pairs in approximately equal amounts in Roche GC RICH kitreactions using the manufacturer's conditions with GC-RICH resolutionbuffer at a concentration of 0.5M in a volume of 25 μl. Reactions werecarried out using the 2 step cycling conditions below:—

Cycle 1-2 94° C. 30 sec 65° C. 30 sec 72° C. 60 sec

This reaction acts as a primer extension producing full length fusions.Oligonucleotide primers were then added to PCR amplify the full lengthmolecules using the cycling conditions below.

Cycle 3-15 Final extension 94° C. 30 sec 65° C. 30 sec 72° C. 60 sec 72°C. 300 sec

When samples of the above SOE reactions were analysed on a 1%agarose/TBE gel and stained with ethidium bromide, both reactions wereshown to contain DNA fragments of approximately 750 by (the expectedsize).

The SOE products were isolated and purified using Geneclean Spin kitsand cloned into pCRTOPO BluntII (Invitrogen). For each SOE product(V_(H)->V_(L) and V_(L)->V_(H)) a TOPO clone was fully characterised byDNA sequencing. pDGF expression constructs (pDGF-SV63-VHVL andpDGF-SV63-VLVH) were then created by the excision of the scFv sequencesfrom pCRTOPOBluntII using NdeI and EcoRI, and subsequent ligation intosimilarly prepared pDGF vector backbone DNA. pIMS147 expressionconstructs (pIMS-SV63-VHVL and pIMS-SV63-VLVH) were also generated byexcision of the scFv sequences from pCRTOPOBluntII using NcoI and EcoRI,and subsequent ligation into similarly prepared pIMS147 vector backboneDNA. Ligation reactions were transformed into E. coli TOP10 cells(Invitrogen) following the manufacturers instructions and transformantsidentified. Correct pIMS147-SV63 scFv and pDGF-SV63 scFv clones wereidentified by DNA sequence analysis to confirm maintenance of readingframe and accuracy of sequence.

2.5.5 Expression of Recombinant scFv Protein in E. coli

pIMSSV63-scFv E. coli TOP10 clones were tested for expression of solublescFv protein following the guidelines described by Charlton (in“Antibody Engineering: methods & protocols” pg 245-254, Ed. B.K.C. Lo,Humana Press, 2004). Briefly, overnight cultures of selected clones wereused to inoculate 500 ml of 2TY(amp/glu) (16 g Bacto-peptone/5 g Yeastextract/5 g NaCl/2% (w/v) glucose in 1 litre, pH 7.5, +100 μg/mlampicillin) in 2.51 flasks, which were then incubated at 37° C. and 250rpm until the OD₆₀₀ reached approximately 0.8. At this point, the cellswere harvested by centrifugation at 3000 g and transferred to 500 mlfresh 2TY(amp/suc) (16 g Bacto-peptone/5 g Yeast extract/5 g NaCl/0.4Msucrose in 1 litre, pH 7.5, +100, μg/ml ampicillin) in 2.5 l flasks.Incubation then commenced at 30° C. and 250 rpm for 1 hour, prior to theaddition of IPTG to a final concentration of 1 mM. Incubation, thenproceeded for a further 16 hours at which point cells and media wereharvested and analysed for the presence of recombinant scFv usingSDS-PAGE and Western blot procedures. Western analysis using theanti-FLAG M2-HRP antibody (Sigma) in combination with BM POD chromogenicsubstrate solution (Roche) indicated good levels of expression ofrecombinant protein (of the expected size molecular weight of approx 30kDa) in both soluble cell lysate and media samples for both orientationsof the scFv.

Recombinant scFv protein was purified using IMAC column chromatographyand tested for functionality by competition with parental SV63 MAb forantigen binding in an immunocytochemical assay using Caco2Bbe1 cells(CRL-2102, American Type Culture Collection). Both orientations of thescFv were observed to inhibit the binding of parental SV63 MAb to thecell surfaces, indicating maintenance of the antigen binding surface inthe engineered scFv.

Example 3 Antibody Purification 3.1 Preparation of Peptide AffinityColumns

For the purification of peptide-specific antibodies from polyclonalsera, peptide affinity columns are produced. Depending on the N-terminalresidue of the peptide, one of two methods for column preparation wasused. These used either SulfoLink or AminoLink coupling gel as theaffinity matrix.

SulfoLink coupling gel (Perbio) allows covalent immobilisation ofsulphydryl-containing peptides to an agarose gel support for use inaffinity purification procedures. RSPEs are coupled to this gel, using aprotocol supplied by the manufacturer and summarised below.

10 ml of SulfoLink gel slurry (5 ml gel bed volume) is equilibrated toroom temperature. The gel slurry is poured into a column andequilibrated with 20 ml of coupling buffer (50 mM Tris, 5 mM EDTA pH8.5) (Sigma). 1 mg of synthetic peptide is dissolved in 5 ml of couplingbuffer and added to the column. The column is sealed and incubated atroom temperature with mixing by inversion for 15 minutes, then the gelis allowed to settle for 30 minutes. Excess buffer is allowed to drain,before the column is washed with 15 ml of coupling buffer. Non-specificbinding sites are blocked with 5 ml of 50 mM L-cysteine hydrochloride(Sigma) in coupling buffer. The column is sealed and incubated with andwithout mixing, as described above. The column is washed with 30 ml of1M sodium chloride (Sigma) and prepared for storage by applying 10 ml ofdegassed PBS pH 7.2 containing 0.05% sodium azide (Sigma). The coupledcolumn is stored at 4° C.

AminoLink coupling gel (Perbio) allows covalent immobilisation ofpeptides via the primary amine to an agarose gel support for use inaffinity purification procedures. RSPEs are coupled to this gel using aprotocol supplied by the manufacturer and summarised below.

10 ml of AminoLink gel slurry (5 ml gel bed volume) is equilibrated toroom temperature. The gel slurry is poured into the column andequilibrated with 20 ml of coupling buffer (0.1M sodium phosphate, pH7.5, 0.05% sodium azide. 1 mg of synthetic peptide is dissolved in 5 mlof coupling buffer. 250 μl of 1M sodium cyanoborohydride made up in0.01M sodium hydroxide (Sigma) is then added to the peptide solution,which is poured into the column. The column is sealed and incubated atroom temperature with mixing by inversion for 6 hours. The column isallowed to stand and then the supernatant is removed. 5 ml of 1MTris-HCl pH 7.4 (Sigma) and 250 μl of 1M sodium cyanoborohydride made upin 0.01M sodium hydroxide is added. The column is sealed and incubatedat room temperature with mixing by inversion for 30 minutes. The columnis washed with 30 ml of 1M sodium chloride and prepared for storage byapplying 10 ml of degassed PBS containing 0.05% sodium azide. Thecoupled column is stored at 4° C.

3.2 Purification of Peptide-Specific Antibodies from Polyclonal Sera

Polyclonal rabbit sera are centrifuged at 2500×g for 10 minutes,filtered using 0.45 micron membranes and then diluted 1:1 with PBS. Thepeptide affinity column is allowed to reach room temperature and thenequilibrated with 4 column volumes of PBS. A prepared serum solution isadded to the column with the flow through being reapplied to the column.The column is washed with 6 column volumes of PBS. Peptide-specificantibody is eluted by applying 0.1M glycine-HCl pH 3.0 (Sigma) to thecolumn and collecting 1 ml fractions in tubes containing 0.1 ml of 1MTris-HCl pH 8.0. Fractions containing protein are then identified byabsorbance at 280 nm and pooled. Purified antibody is dialysed into PBSusing a Slide-a-Lyser cassette with 10,000 molecular weight cut-off(Perbio) and stored at −20° C. Meanwhile, the column is regenerated with3 column volumes of 0.1M glycine-HCl pH 2.5, followed by 8 columnvolumes of PBS.

3.2 Purification of Monoclonal Antibodies

Monoclonal IgG is purified from culture supernatant by protein Gaffinity chromatography using a HiTrap protein G HP 1 ml column (GEHealthcare). Culture supernatant is centrifuged at 2500×g for 10 minutesand filtered using 0.45 micron membranes before use. The maximum flowrate is 1 ml/minute throughout. The column is equilibrated with 10column volumes of PBS pH 7 and the sample is loaded. The column iswashed with 10 column volumes of PBS. Antibody is eluted by applying0.1M glycine-HCl pH 3.0 (Sigma) to the column and collecting 1 mlfractions in tubes containing 0.1 ml of 1M Tris-HCl pH 8.0. Fractionscontaining protein are then identified by absorbance at 280 nm andpooled. Purified antibody is dialysed into PBS using a Slide-a-Lysercassette with 10,000 molecular weight cut-off (Perbio) and stored at−20° C. Meanwhile the column is regenerated with 10 column volumes of0.1M glycine pH 2.5, washed with 10 column volumes of PBS and stored in20% ethanol at 4° C.

Example 4 Antibody Characterisation 4.1 Titration of Anti-PeptideAntibodies by Enzyme-Linked Immunosorbent Assay (ELISA)

Sera obtained from rabbits, mice or hamsters immunised with peptideconjugates are assayed by enzyme-linked immunosorbent assay (ELISA) todetermine the relative magnitude of the antibody responses, based on themethods described by Engvall E., and Perlmann P. Immunochemistry 8,871-874 (1971); Harlow E. et al., Antibodies-A Laboratory Manual, ColdSpring Harbor Laboratory, 1988 pp. 182-183.

Peptide solutions at 2 μg/ml in 35 mM sodium bicarbonate, 15 mM sodiumcarbonate pH 9.5 are added at 100 μl/well to 96-well microtitre platesand incubated for at least four hours at 4° C. Plates are then washedthree times with PBS, 0.05% Tween 20 (PBST) (Sigma). Remaining bindingsites are blocked with 200 μl well of PBS containing 1% skimmed milkpowder (Marvel, Premier Foods, St Albans) for 30 minutes at roomtemperature. After washing as above, PBST is added to the plates at 100ul/well. Initial dilutions of sera are added to duplicate wells incolumn 1 and then double-diluted across the wells of the plates, leavingthe wells in column 12 containing PBST only. The plates are incubated atroom temperature for 2 hours. After further washes as above, the platesare incubated at room temperature for 1 hour with the appropriateanti-species conjugated antibody diluted to 1/10,000 in PBST. Rabbitsera samples are incubated with goat anti-rabbit IgG horseradishperoxidase (HRP) conjugate (Sigma); hamster sera with rabbit anti-Syrianhamster IgG HRP conjugate (Stratec, Soham, Cambridgeshire) and mousesera samples will be incubated with both rabbit anti-mouse IgG HRPconjugate (Sigma) and goat anti-mouse IgG Fc fragment HRP conjugate(Sigma). Substrate solution is prepared by diluting one tabletcontaining 1 mg of 3,3′,5,5′ tetramethylbenzidine dihydrochloride(Sigma) in 10 ml of 24 mM citric acid, 60 mM sodium phosphate pH 5.0 andadding 2 μl of 30% solution of hydrogen peroxide (Sigma). After furtherwashing, fresh substrate solution is added to the plates at 100 ul/welland the plates are left in the dark at room temperature for 30 minutes.The resulting colour development is stopped by adding 50 ul/well of 3Msulphuric acid. The optical density of the plate is then read at 450 nmusing a microtitre plate reader.

4.2 ELISA Results

4.2.1 Polyclonal Anti-Sera from Rabbits

Sera taken post-third dose from rabbits immunised with RSPEs from thefollowing target proteins (see Example 2.1 above) Rat oligopeptidetransporter PepT1, Rat CD155 (PVR-poliovirus receptor; Tage4), Rat GTR2,Rat CFTR, Rat CNT2, Rat MDR1, Mouse MDR1, Rat sucrase-isomaltase, MouseGLUT 7, Rat GTR5, Rat OATP-B, Rat GCC, Rat PLB, Rat LPH, Rat AMPN, RatMCDL, and Rat SCAB were assayed by peptide ELISA as described in Example4.1 above. Test and pre-immune sera from each rabbit were assayed bytitration against their immunising peptide. The immune response wasassessed by calculating 50% binding titre values (dilution required toreduce maximum signal to 50%). The results are summarised in Table 6below.

TABLE 6 ELISA results showing that rabbit polyclonal antibodies againstthe specified RSPEs have been generated. 50% Binding Titre SOURCEPROTEIN Immunising RSPE Serum (fold dilution) Rat oligopeptide SEQ IDNO: 1 Rabbit 1 (pre-immune) — transporter PepT1 Rabbit 1(test) >500,000  Rabbit 2 (pre-immune) — Rabbit 2 (test) >500,000  Ratoligopeptide SEQ ID NO: 2 Rabbit 1 (pre-immune) — transporter PepT1Rabbit 1 (test) 33,000 Rabbit 2 (pre-immune) — Rabbit 2 (test)   5000Rat CD155 (PVR, SEQ ID NO: 3 Rabbit 1 (pre-immune) — Tage4) Rabbit 1(test) 15,000 Rabbit 2 (pre-immune) — Rabbit 2 (test)  5,000 Rat GTR2(GLUT2) SEQ ID NO: 4 Rabbit 1 (pre-immune) — glucose transporter Rabbit1 (test) 55,000 Rabbit 2 (pre-immune) — Rabbit 2 (test) 40,000 Rat CFTRchloride SEQ ID NO: 5 Rabbit 1 (pre-immune) — transporter Rabbit 1(test) 95,000 Rabbit 2 (pre-immune) — Rabbit 2 (test) 44,000 Rat CNT2nucleoside SEQ ID NO: 6 Rabbit 1 (pre-immune) — transporter Rabbit 1(test) 50,000 Rabbit 2 (pre-immune) — Rabbit 2 (test) 50,000 Rat MDR1multidrug SEQ ID NO: 8 Rabbit 1 (pre-immune) — resistance transporterRabbit 1 (test) 35,000 Rabbit 2 (pre-immune) — Rabbit 2 (test) 64,000Mouse MDR1 multidrug SEQ ID NO: 9 Rabbit 1 (pre-immune) — resistancetransporter Rabbit 1 (test) 41,000 Rabbit 2 (pre-immune) — Rabbit 2(test)  8,000 Rat sucrase-isomaltase SEQ ID NO: 10 Rabbit 1 (pre-immune)— Rabbit 1 (test) 31,000 Rabbit 2 (pre-immune) — Rabbit 2 (test)  7,000Mouse GLUT7 glucose SEQ ID NO: 11 Rabbit 1 (pre-immune) — transporterRabbit 1 (test) 11,000 Rabbit 2 (pre-immune) — Rabbit 2 (test) 34,000Mouse GLUT7/Rat SEQ ID NO: 12 Rabbit 1 (pre-immune) — GTR5 (GLUT5)glucose Rabbit 1 (test) 30,000 transporters Rabbit 2 (pre-immune) —Rabbit 2 (test) 34,000 Rat OATP-B SEQ ID NO: 15 Rabbit 1 (pre-immune) —(SLC21A9) organic Rabbit 1 (test) 66,000 anion transporting Rabbit 2(pre-immune) — polypeptide Rabbit 2 (test) 12,000 Rat GCC (guanylyl SEQID NO: 27 pre-immune — cyclase) test 90,000 Rat PLB SEQ ID NO: 28pre-immune — (phospholipase B) test 170,000  Rat LPH (lactase- SEQ IDNO: 29 pre-immune — phlorizin hydrolase) test 35,000 Rat AMPN SEQ ID NO:31 pre-immune — (aminopeptidase N) test 220,000  Rat MCDL (mucin and SEQID NO: 32 pre-immune — cadherin-like protein) test 16,000 Rat SCAB(amiloride- SEQ ID NO: 34 pre-immune — sensitive sodium test 18,000channel beta-subunit)

Sera from each of the test bleeds was affinity purified as described inExample 3.2 and then tested by ELISA as described previously. Theresults are given in Table 7 below.

TABLE 7 ELISA results for affinity-purified antibodies obtained fromrabbit test sera. Elution Concentration 50% Binding Immunising SerumVolume of Eluate Titre (fold RSPE sample (ml) (mg/ml) dilution) SEQ IDNO: 1 Rabbit 1 10 0.47 1 × 10⁷ Rabbit 2 10 0.47 930,000 SEQ ID NO: 2Rabbit 1 3.5 0.08 32,000 Rabbit 2 2 0.12 3,000 SEQ ID NO: 3 Rabbit 1 30.25 8,000 Rabbit 2 3.5 0.26 6,000 SEQ ID NO: 4 Rabbit 1 7.25 0.1418,000 Rabbit 2 4 0.21 5,000 SEQ ID NO: 5 Rabbit 1 5.5 0.72 51,000Rabbit 2 4.5 0.32 19,000 SEQ ID NO: 6 Rabbit 1 7 0.52 47,000 Rabbit 2 60.35 16,000 SEQ ID NO: 8 Rabbit 1 5.5 0.64 42,000 Rabbit 2 9 0.65 31,000SEQ ID NO: 9 Rabbit 1 5 0.26 33,000 Rabbit 2 3.25 0.20 8,000 SEQ ID NO:10 Rabbit 1 6 0.35 16,000 Rabbit 2 4.5 0.16 2,000 SEQ ID NO: 11 Rabbit 15.5 0.66 7,000 Rabbit 2 9 0.94 7,000 SEQ ID NO: 12 Rabbit 1 8 0.2330,000 Rabbit 2 5 0.09 34,000 SEQ ID NO: 15 Rabbit 1 9.5 0.31 66,000Rabbit 2 6 0.19 12,000 SEQ ID NO: 27 Rabbit 1 6 0.29 20,000 SEQ ID NO:28 Rabbit 1 6 0.66 19,000 SEQ ID NO: 29 Rabbit 1 6 0.20 12,000 SEQ IDNO: 31 Rabbit 1 7 0.18 10,000 SEQ ID NO: 32 Rabbit 1 3 0.16 8,000 SEQ IDNO: 34 Rabbit 1 5 0.18 5,000

Harvest bleeds for rabbits immunised with the RSPE from the Rat CNT2nucleoside transporter were also affinity purified and tested by ELISA.The harvest bleed from rabbit 1 was eluted in 36.5 ml at a concentrationof 0.86 mg/ml and the 50% binding titre was assayed at 1 in 47,000. Theharvest bleed from rabbit 2 was eluted in 27.5 ml at a concentration of0.57 mg/ml and the 50% binding titre was assayed at 1 in 44,000.

The affinity purified polyclonal antibodies from rabbit 1 were furtherpurified using a Protein A column (Amersham), dialysed against phosphatebuffered saline (PBS) and tested by ELISA. This resulted in 13 mls offurther purified polyclonal IgG against the CNT2 derived RSPE (SEQ IDNO: 6), which had a concentration of 1.97 mg/ml and a 50% binding titreof 1 in 90,000.

4.2.2 Polyclonal Anti-Sera from MiceSera taken post-third dose from mice immunised with RSPEs from thefollowing target proteins (see Example 2.2 above), Rat oligopeptidetransporter PepT1, Rat CD155 (PVR-poliovirus receptor; Tage4), Rat GTR2,Rat CFTR, Rat CNT2, Rat MDR1, Mouse MDR1, Rat sucrase-isomaltase, MouseGLUT 7, Rat GCC, Rat PLB, and Rat LPH, were assayed by peptide ELISA asdescribed in Example 4.1 above. Test serum from each mouse was assayedby titration against the immunising peptide. The immune response wasassessed by calculating 50% binding titre values (dilution required toreduce maximum signal to 50%). The results are summarised in Table 8below.

TABLE 8 ELISA results showing that mouse polyclonal antibodies againstthe specified RSPEs have been generated. Immunising Mouse serum 50%Binding Titre SOURCE PROTEIN RSPE sample (fold dilution) Ratoligopeptide transporter SEQ ID NO: 1 1 >100,000   PepT1 2 70,000  370,000  4 >100,000   Rat oligopeptide transporter SEQ ID NO: 2 1 — PepT12 10,000  3 — 4 — Rat CD155 (PVR, Tage4) SEQ ID NO: 3 1 3,000 2 62,000 3 41,000  4 — Rat GTR2 (GLUT2) glucose SEQ ID NO: 4 1 2,000 transporter2 2,000 3 — 4 — 5 — 6 7,000 7 7,000 8 4,000 Rat CFTR chloridetransporter SEQ ID NO: 5 1 40,000  2 40,000  3 26,000  4 3,000 5 — 6 — 7 2000 8 — Rat CNT2 nucleoside transporter SEQ ID NO: 6 1 20,000  2 3,5003 19,000  4 60,000  5 — 6 2,000 7 1,500 8 2,000 9 2,000 10 2,000 1132,000  12 15,000  Rat MDR1 multidrug resistance SEQ ID NO: 8 1 1,000transporter 2 18,000  3 77,000  4 — Mouse MDR1 multidrug SEQ ID NO: 9 112,000  resistance transporter 2 12,000  3 4,000 4 4,000 Ratsucrase-isomaltase SEQ ID NO: 10 1 3,000 2 7,000 3 7,000 4 2,000 MouseGLUT7 glucose SEQ ID NO: 11 1 4,000 transporter 2 2,000 3 1,000 4 2,0005 — 6 — 7 — 8 — Rat GCC (guanylyl cyclase) SEQ ID NO: 27 1 2,500 2 — 32,000 4 — 5 2,000 6 2,000 7 6,000 8 2,000 Rat PLB (phospholipase B) SEQID NO: 28 1 1,500 2 — 3 1,000 4 — 5 — 6 — 7 — 8 — Rat LPH(lactase-phlorizin SEQ ID NO: 29 1 1,500 hydrolase) 2 3,000 3 3,000 412,000  5 15,000  6 15,000  7 15,000  8 15,000 4.2.3 Monoclonal Antibodies to an RSPE from the Rat OligopeptideTransporter PepT1

40 ml of culture supernatant from each of the seven positive monoclonalcell lines producing antibodies against the RSPE having SEQ ID NO 1 (seeExample 2.3 above) was affinity purified as described in Example 3.2,and then tested by ELISA as described in Example 4.1 above. Purifiedmonoclonal antibodies were assayed by titration against the immunisingpeptide (SEQ ID NO: 1). The immune response was assessed by calculating50% binding titre values (dilution required to reduce maximum signal to50%). The results are summarised in Table 9 below.

TABLE 9 ELISA results for affinity purified culture supernatant fromhybridoma cell lines generated from mice immunised with an RSPE from theRat oligopeptide transporter PepT1. Elution 50% Hybridoma VolumeConcentration Binding Titre Cell Line (ml) (mg/ml) (fold dilution)1645.142.002 3 1.4 300,000  1644.112.040 4 1.1 12,500 1645.451.209 2 0.1— 1647.372.245 3 1.2 92,000 1646.461.144 2 0.08 — 1647.449.047 2 0.09 —1646.240.206 2 0.1 —

Hybridoma cell lines 1645.142.002, 1644.112.040 and 1647.372.245 clearlyproduce monoclonal antibodies which recognise the RSPE having thesequence of SEQ ID NO 1, which is derived from Rat PepT1.

4.2.4 Monoclonal Antibodies to a RSPE from the Mouse GLUT7 Transporter

40 ml of culture supernatant from each of the four positive monoclonalcell lines producing antibodies against the RSPE having SEQ ID NO 11(see Example 2.3 above) was affinity purified as described in Example3.2, and then tested by ELISA as described in Example 4.1 above.Purified monoclonal antibodies were assayed by titration against theimmunising peptide (SEQ ID NO: 11). The immune response was assessed bycalculating 50% binding titre values (dilution required to reducemaximum signal to 50%). The results for two out of the four anti-GLUT7monoclonal antibodies are summarised in Table 10 below.

TABLE 10 ELISA results for affinity purified culture supernatant fromhybridoma cell lines generated from mice immunised with an RSPE from theMouse GLUT7 transporter. Elution 50% Hybridoma Volume ConcentrationBinding Titre Cell Line (ml) (mg/ml) (fold dilution) 1657.183.245.2332.5 0.86 1,500 1657.120.223.282 2.75 0.59 4,000

4.3 Immunohistochemistry of Rodent Gut Tissue Sections

Rat gut tissue was snap frozen in liquid nitrogen and stored at −80° C.until use. All tissue was sectioned on a cryostat at −20° C. and tissuewas placed onto positively charged slides, fixed in ice cold acetone for10 min and left to air dry. Slides were stored at −20° C. and usedwithin 1 month of sectioning. Prior to starting immunohistochemistry,all slides were allowed to warm up to room temperature. They were thenloaded onto the Sequenza, for manual IHC to be performed. All slideswere blocked for endogenous peroxidases by application of peroxidaseblock (provided in Dako Envision® kit) for 6 min at room temperature.The slides were then washed in TRIS-buffered saline (10 mM) with tween20 (0.1%) (TBST) for 5 min. Non-specific proteins were blocked byaddition of 5% milk proteins (Marvel™) for 30 min at room temperature.Slides were washed for 5 min in TBST. Primary antibody (made up in TBSTwith 1% Marvel™, at dilutions stated in table) was applied and slideswere incubated for 1 hour at room temperature. Slides were then washedin TBST (2×5 min). Envision® rabbit polymer was applied and incubated onslides for 30 min at room temperature. Slides were washed (2×5 min) inTBST. DAB was applied for 5 min at room temperature. Slides were washedin distilled H₂O, dehydrated through graded alcohols and xylene and thenmounted in DPX.

TABLE 11 Summary of immunohistochemistry data for rabbit polyclonalantibodies raised against specific RSPEs SEQ ID NO of RPSE againstDilution which of anti- anti- body body where Blocked with was Dilutionstaining blocking raised Source Protein range present Rat Duodenum RatJejunum Rat Ileum Rat Colon peptide 1 Rat oligopeptide 1:20-1:200 1:50Villi epithelial cells Villi epithelial No staining Villi epithelialcells Blocking data transporter (inconsistent) cells (inconsistent)inconclusive PepT1 staining (inconsistent) staining staining 2 Ratoligopeptide 1:10-1:200 No staining No staining transporter PepT1 3 RatCD155 1:10-1:200 Lamina propria Lamina propria (PVR, Tage4) staining(non- staining (non- specific) specific) 4 Rat GTR2 1:10-1:200 1:10Crypt epithelial Crypt epithelial Crypt epithelial Crypt epithelial(GLUT2) cells, patchy villi cells staining cells staining cells stainingglucose epithelial cells transporter staining 5 Rat CFTR 1:25-1:200 1:50Villi epithelial cells Crypt epithelial Crypt epithelial Villiepithelial cells Staining was chloride and crypt cells staining cellsstaining and crypt successfully transporter epithelial cells epithelialcells blocked with staining staining excess peptide 6 Rat CNT21:25-1:200 1:50 Muscularis Muscularis Muscularis Muscularis Staining wasnucleoside mucosa staining mucosa staining mucosa staining mucosastaining successfully transporter and villi and crypt and villi andcrypt and villi and crypt blocked with epithelial cells epithelial cellsepithelial cells excess peptide staining staining staining 8 Rat MDR11:10-1:200 No staining No staining multidrug resistance transporter 9Mouse MDR1 1:10-1:200 No staining No staining multidrug resistancetransporter 10 Rat sucrase- 1:10-1:200 No staining No stainingisomaltase 11 Mouse GLUT7 1:25-1:200 1:50 Staining on Crypt epithelialCrypt epithelial Crypt epithelial Staining was glucose Brunners Glandscells staining cells staining cells staining successfully transporterblocked with excess peptide 12 Mouse 1:10-1:200 No staining No stainingGLUT7/Rat GTR5 (GLUT5) glucose transporters 15 Rat OATP-B 1:10-1:200Possible crypt (SLC21A9) epithelial staining organic anion transportingpolypeptide 27 Rat GCC 1:25-1:200 1:25 Crypt epithelial Crypt epithelialCrypt epithelial Crypt epithelial Staining was (guanylyl cells and villicells and villi cells and villi cells and villi successfully cyclase)epithelial cells epithelial cells epithelial cells epithelial cellsblocked with staining staining staining staining excess peptide 28 RatPLB 1:25-1:200 1:50 Villi and crypt Villi and crypt Villi and cryptVilli and crypt Staining was (Phospholipase epithelial cells epithelialcells epithelial cells epithelial cells not B) staining stainingstaining staining successfully blocked with excess peptide - indicatingnon-specific staining seen 29 Rat LPH 1:25-1:200 1:25 Possible cryptPossible crypt No staining No staining Staining in (lactase- and villiepithelial and villi epithelial duodenum and phlorizin cells stainingcells staining jejunum hydrolase) successfully blocked with excesspeptide 31 Rat AMPN 1:25-1:200 No staining No staining No staining Nostaining (aminopeptidase N) 32 Rat MCDL 1:25-1:200 No staining Nostaining No staining No staining (mucin and cadherin-like protein) 34Rat SCAB 1:25-1:200 No staining No staining No staining No staining(amiloride- sensitive sodium channel beta- subunit)

Specific staining was assessed with the use of blocking peptides.Primary antibodies, at the dilution to give optimum staining, wereincubated with a 10 fold excess of peptide for 2 hours at roomtemperature in TBS with 1% Marvel™, before application onto the slidesthe remaining protocol was as described above. The results aresummarised in Table 11 above.

Example 5 Production of Recombinant Protein Toxins 5.1 RecombinantGelonin Production

The gene encoding the 251 amino acid gelonin protein (see Nolan et al.,1993, Gene 134: 223-227) together with the pelB leader sequence and aC-terminal hexa-histidine tag were codon optimised for E. coliexpression and cloned into an E. coli expression vector (pDGF—derivedfrom the NEB vector pMALc-2, pDGF contains all of the features ofpMALc-2 except for the sequence encoding the maltose binding protein,which has been excised) under the control of the hybrid tac promoter.The recombinant vector was transformed into E. coli strain TOP10.

Expression of gelonin was achieved by growing the transformed TOP10cells in LB medium with induction at 28° C. with 1 mM IPTG for 20 hours.Following harvesting of E. coli cells the cells were disrupted using aFrench pressure cell (Constant Systems; disruption at 20,000 psi) in1×PBS/30 mM Imidazole. The soluble extract was applied to a GraviTrapcolumn (GE Healthcare) and after a 15 ml wash with 1×PBS/30 mMImidazole, gelonin was eluted in 1×PBS/500 mM Imidazole. As a secondpurification step, the eluate was desalted into 20 mM sodium phosphatebuffer pH 8.0 and applied to a Resource S cation exchange column with a0 to 1M salt gradient elution.

To determine whether the recombinant gelonin was active (functional) thepurified gelonin protein was tested in a protein translation inhibitionassay (TNT T7 quick coupled transcription/translation system, Promega)using T7 luciferase DNA as the substrate. As a positive control,cycloheximide was used. This demonstrated that the recombinant purifiedgelonin inhibited translation of the T7 luciferase DNA.

5.2 Recombinant VIP2A Production

The gene encoding the 464 amino acid VIP2A protein (see U.S. Pat. No.5,849,870) together with a C-terminal hexa-histidine tag was codonoptimised for E. coli expression and cloned into the pET24a expressionvector (Novagen). The recombinant vector was transformed into E. colistrain BL21(DE3).

Expression of VIP2A was achieved by growing the transformed BL21(DE3)cells in LB medium with induction at 28° C. with 1 mM IPTG for 20 hours.Following harvesting, the cells were disrupted using a French pressurecell as described in Example 5.1 above. The soluble extract was appliedto a HisTrapHP column (GE Healthcare) and after washing with 5 columnvolumes of PBS/20 mM imidazole, VIP2A protein was eluted with a gradientof up to 500 mM imidazole.

SDS-PAGE analysis of the soluble extract and sample from thepurification revealed a band at the expected size for recombinantlyproduced Histidine-tagged VIP2A.

5.3 Expression Construct for the Production of Recombinant Granzyme B

A gene encoding the 228 amino acid mature Granzyme B protein from rat(see Genbank accession No M34097 for sequence information) wasartificially synthesised (DNA fragment 050031) and cloned intopCR-Script to give plasmid p050031. A nested PCR approach was taken tointroduce the Granzyme B coding sequence into the expression vectorpET32a(+) (Novagen).

The mature Granzyme B coding sequence from p050031 was PCR amplifiedusing primers RoPro070 petEK/rGrzB F1 (SEQ ID NO: 51) and RoPro067 rGrzBR (SEQ ID NO: 52) to introduce a 5′ tail homologous to the enterokinasesite present in the fusion tag in pET32a(+). The resulting PCR productwas used as a template for the second part of the nested PCR, which wascarried out using primers RoPro076 petEK/rGrzB F2 (SEQ ID NO: 53) andRoPro067rGrzB R (SEQ ID NO: 52) to introduce a 5′ KpnI site.

The final PCR product was digested with KpnI/NotI and ligated intosimilarly digested pET32a(+) to give expression vector pET32a(+)::rGrzB.This results in a clean fusion between the N-terminal expression tagfrom the host vector and recombinant Granzyme B coding sequence, thuspermitting activation of recombinant Granzyme B by treatment withenterokinase following expression. The final expression vectorpET32a(+)::rGrzB may be transformed into any suitable E. coli expressionhost (e.g. RosettaGami (DE3)).

The sequences of the primers used in the construction ofpET32a(+)::rGrzB are as follows (all primer sequences are given 5′ to3′): RoPro070 petEK/rGrzB F1 (SEQ ID NO: 51)GGTACCGACGACGACGACAAGATCATCGGTGGTCACGAAGCT AAGCCAC; RoPro067 rGrzB R(SEQ ID NO: 52) AGCTGGCGGCCGCCTAGGAC; RoPro076 petEK/rGrzB F2 (SEQ IDNO: 53) AGATCTGGGTACCGACGACGACGA C.

Example 6 Conjugation of Antibody Components to Toxins 6.1 Conjugationof Commercially Available Gelonin to Polyclonal Anti-Rat CNT2 Antibodies

The strategy chosen to carry out this procedure is to activate theantibody with the cross linker SPDP (N-succinimidyl3-[2-pyridyldithio]propionate; Pierce Chemical Co.) which will formdisulphide link to thiolated toxin. The method used came from; Hermanson1996 “Bioconjugate Techniques” (Academic Press pp. 509) which statedthat the conjugation would not interfere with the activity of the toxingelonin (30 kDa). Both 5:1 and 10:1 molar ratios of gelonin to anti-CNT2polyclonal antibody were used to obtain the most efficient reaction mix.

6.1.1 SPDP Treatment of CNT2 Polyclonal Antibody

A 1 ml aliquot of the protein A purified rabbit 1 polyclonal IgG (seeExample 4.2.1, harvest bleed from rabbit 1 was affinity purified andthen purified using a Protein A column) was concentrated using acentricon spin concentrator with 10 kDa cut off. The resulting 160 ulwas made up to a 10 mg/ml solution in PBS 10 mM EDTA pH 8. 6 ul SPDP (3mg/ml in DMF) was added to 200 ul of antibody solution and incubated for30 mins at room temperature. The reaction mixture was applied to a PD10desalting column equilibrated with PBS+10 mM EDTA pH 8. All of the 3.5ml sample volume was collected and concentrated using a centriconconcentrator to obtain SPDP-treated anti-CNT2 antibody at a finalconcentration of 3.6 mg/ml.

6.1.2 Thiolation of Gelonin

Gelonin was obtained from Aczon SpA as a 5 mg sample of lyophilisedprotein purified from the seeds of Gelonium multiflorum. The sample wasoriginally dissolved in PBS at a concentration of 5 mg/ml. 300 ul ofthis solution was concentrated in a centricon concentrator and thevolume reduced to 55 ul. This was then diluted in 50 mM triethanolamine10 mM EDTA pH 8 to give a 10 mg/ml gelonin solution. 2-Immunothiolanewas dissolved to give a 20 mg/ml solution in distilled water. 10.5 ul of2-immunothiolane solution this was added to 150 ul gelonin solution andthe mixture incubated on ice for a hour. The activated gelonin wasapplied to a PD10 desalting column equilibrated with PBS+10 mM EDTA pH8. All of the 3.5 ml sample volume was collected and concentrated in acentricon concentrator to obtain thiolated gelonin at a finalconcentration of 3 mg/ml.

6.1.3 Conjugation of Anti-CNT2 Polyclonal Antibody to Gelonin

To obtain a 5:1 molar ratio of gelonin to anti-CNT2 antibody, 0.5 mgthiolated gelonin needed to be reacted with 0.5 mg anti-CNT2 antibody.Accordingly, 138 ul of SPDP treated anti-CNT2 antibody was added to 167ul of thiolated gelonin.

The reaction was also carried out at a 10:1 molar ratio of gelonin toanti-CNT2 antibody (1 mg thiolated gelonin in 333 ul was added to 0.5 mgSPDP-treated anti-CNT2 antibody in 138 ul). Each reaction was sealedunder nitrogen and incubated for 20 hours at 4° C. After this time anyunreacted sulfhydryl residues were blocked by the addition ofiodoacetamide to a final concentration of 2 mM.

6.1.4 Analysis of Conjugate

Each reaction mixture was analysed by MALDI-TOF mass spectrometry,SDS-PAGE and ELISA. Data (not shown) obtained from SDS-PAGE separationof the reaction mixtures and mass spectrometry revealed show that eachconjugation reaction was successful, and species were identified with 1,2 and 3 molecules of gelonin conjugated to a single antibody molecule.Although mass spectrometry data revealed the presence of someunconjugated antibody, the amount was insufficient to be observed on acoomassie stained SDS-PAGE gel. There appeared to be no difference inefficiency of conjugation between the 2 reaction ratios.

ELISA assays were carried out as described previously on each reactionmixture in order to assess whether the binding activity of the antibodywas affected by the conjugation reaction. The data obtained issummarised in Table 12 below.

TABLE 12 ELISA data for conjugation of gelonin to anti-CNT2 polyclonalantibodies Sample 50% Binding Titre Anti-CNT2 polyclonal 0.051 ug/mlSPDP-treated anti-CNT2 polyclonal 0.134 ug/ml 5:1 molar ratio thiolatedgelonin:SPDP  0.33 ug/ml treated anti-CNT2 polyclonal 10:1 molar ratiothiolated gelonin:SPDP  0.22 ug/ml treated anti-CNT2 polyclonal

It can be that the binding has been affected by both the addition of theSPDP polylinker and by the conjugation to gelonin. However, theconjugated antibody reaction mixture still exhibits significant binding.This can be attributed to both conjugated and non-conjugated antibody inthe reaction mixture. Since the amount of unconjugated antibody is low,it is assumed that a good proportion of the binding observed may beattributed to the conjugated antibody.

6.2 Conjugation of Recombinant Gelonin to Polyclonal Anti-Rat CNT2Antibodies

The same strategy as outlined above in Example 6.1 was adopted in orderto conjugate recombinant gelonin, produced as described in Example 5above, to polyclonal anti-Rat CNT2 antibodies. A 5:1 gelonin toanti-CNT2 polyclonal antibody molar ratio was used to obtain the mostefficient reaction mix.

6.2.1 SPDP Treatment of Anti-CNT2 Polyclonal Antibody

3.75 mg of the protein A purified rabbit 1 polyclonal as a 10 mg/mlsolution in PBS/10 mM EDTA pH 8 was mixed with 11.25 ul SPDP (3 mg/ml inDMF) and incubated for 30 mins at room temperature. The reaction mixturewas passed through a Zeba (Pierce Chemical Co.) desalting columnpre-equilibrated with PBS/10 mM EDTA pH 8.

6.2.2 Thiolation of Recombinant Gelonin

3.75 mg of recombinant gelonin was made up to 10 mg/ml in 50 mMtriethanolamine 10 mM EDTA pH 8. 2-Iminothiolane (Traut's reagent;Sigma-Aldrich) was dissolved to 20 mg/ml in de-gassed andnitrogen-bubbled deionised H₂O, 26.25 ul of this was added to thegelonin solution and incubated in a nitrogen atmosphere on ice for onehour. The thiolated gelonin was passed through a Zeba (Pierce ChemicalCo.) desalting column pre-equilibrated with PBS/10 mM EDTA pH 8.

6.2.3 Conjugation of Anti-CNT2 Polyclonal Antibody to RecombinantGelonin

The SPDP-reacted antibody solution was mixed with the thiolatedrecombinant gelonin solution resulting in equal quantities of eachprotein constituent, providing a 5:1 molar ratio of recombinant geloninto antibody. The reaction was sealed under nitrogen and incubated for 20hours at 4° C.

After this time any unreacted sulfhydryl groups were blocked by theaddition of iodoacetamide to a final concentration of 2 mM, followed bya minimum of one hours incubation at room temperature.

6.2.4 Separation of the Conjugate from Unconjugated Recombinant Gelonin

Gel-filtration with PBS was used to separate unconjugated geloninmolecules from conjugates. A 10/300 Superdex 200 column(Amersham-Pharmacia) was equilibrated in PBS for 3 column volumes (CVs)using an Akta FPLC unit (Amersham-Pharmacia). Chromatography wascontrolled by a PC running Unicorn software (Amersham-Pharmacia), whichinjected the sample and then maintained a 0.5 ml/min flow rate, with 0.5ml fractions collected into a 96 well block after 0.1 CVs had eluted.Elution was allowed to proceed for 1.4 CVs.

Selected fractions were analysed on a non-reducing SDS-PAGE gel (4-12%bis-tris NuPage in MOPS buffer; Invitrogen) as described by themanufacturer. The gel was stained using the SimplyBlue coomassie stain(Invitrogen) as described by the manufacturer. The fractions that weredetermined to contain the conjugates were pooled and submitted to thenext step of purification.

6.2.5 Separation of the Conjugate from Unconjugated Anti-CNT2 Antibody

The presence of a hexa-histidine C-terminal tail on the recombinantgelonin molecules allowed the application of immobilised metal-chelateaffinity chromatography (IMAC) as a second chromatographic step in thepurification of the conjugates. The lack of a hexa-histidine motif onthe unconjugated antibody (still present in the conjugate fractionsafter gel-filtration) allows the removal of free antibody fromhexa-histidine containing conjugates. Therefore, the pooled fractionsobtained in 6.2.4 above were passed through a HisTrap HP 1 mlnickel-affinity column (pre-equilibrated by 5 CVs of PBS/15 mMimidazole; Amersham Biosciences) attached to an Akta FPLC unitcontrolled by a PC running Unicorn software (Amersham Biosciences).Loading and washing of the column was carried out over 5 CVs usingPBS/15 mM imidazole as the load/wash buffer. Elution of hexa-histidinecontaining proteins was effected by the application of a gradient of 15mM to 500 mM imidazole (in PBS) over 20 CVs. Fractions were collected in0.5 ml volumes in a 96 well block.

Selected fractions were analysed on a non-reducing SDS-PAGE gel (4-12%bis-tris NuPage in MOPS buffer; Invitrogen) as described by themanufacturer. The gel was stained using the SimplyBlue™ coomassie stain(Invitrogen) as described by the manufacturer.

The gel revealed that the conjugates were substantially purified awayfrom free antibody by the IMAC step. Fractions containing the conjugateswere pooled and desalted into PBS using VivaSpin 20 ml spinconcentrators with a 10 kD molecular weight cut-off.

6.2.6 Analysis of Conjugate

MALDI-TOF mass-spectrometry was used to analyse the composition of thepooled conjugate sample relative to unconjugated anti-CNT2 antibody. Themass spectra obtained (not shown), indicated the presence of 1:1, 1:2and 1:3 ratio's of antibody:gelonin conjugate species in the pooledconjugate fraction (Pool B). No singly charged unconjugated antibodyspecies was observed, however a peak on the spectrum was identifiedwhich correlates with a doubly-charged species of unconjugated antibody.Thus, even though the majority of the unconjugated antibody was purifiedaway from conjugated antibody (as indicated by SDS-PAGE analysis of theIMAC purification), it may be inferred that a very small amount ofunconjugated antibody remains in the pooled purified sample.

The functional activity of the conjugate in the MAC-purified pooledsample was assessed by ELISA (to determine the affect of conjugation onantibody binding) and by an in vitro translation inhibition assay (todetermine whether the functional activity of gelonin had been affectedby conjugation).

The ELISA (data not shown) revealed similar results to those observedfor conjugation to commercially supplied gelonin (Example 6.1.4 above):antibody binding was affected by both the addition of the SPDPpolylinker and by the conjugation to recombinant gelonin, but theconjugated antibody reaction mixture still exhibited significant bindingto the Rat CNT2 RSPE. Although the mass-spectrometry analysis indicatedthat there may still be some unconjugated antibody remaining even afterthe IMAC purification, the amount of unconjugated antibody in the MACpurified samples will be less than in the conjugation reaction mixturefor the natural gelonin/anti-CNT2 antibody as a consequence of the IMACpurification. Accordingly, the antibody binding observed may beattributed as being mainly due to anti-CNT2 antibody-recombinant geloninconjugate.

The retention of ribosome-inhibiting capability of the recombinantgelonin conjugated to the anti-CNT2 antibody in the pooled conjugatecontaining sample from the IMAC purification was determined using theTNT quick coupled transcription/translation system (Promega) asdescribed by the manufacturer. FIG. 2 shows the results. Thetranslational inhibition exhibited by conjugates in IMAC purified pooledsample (Pool B) is equivalent to that of the original recombinantgelonin (rGelonin). Thus ribosome-inhibitory capability has beenmaintained following conjugation.

6.3 Conjugation of Commercially Available β-Purothionin to PolyclonalAnti-Rat CNT2 Antibodies

The strategy chosen for this conjugation was to use the TFCS crosslinker (Pierce), which has a large spacer arm, in order to give as muchexposure as possible to the relatively small (5 kDa) β-purothioninmolecule. TFCS has a NHS ester group at one end, which binds to aminegroups on the antibody, and a protected amine group at the other end,which is exposed for reacting to appropriately treated β-purothionin byraising the pH to 8. Carboxyl groups on the purothionin are reacted withEDC to form an unstable amine reactive intermediate which is thenreacted with sulfo-NHS to provide a more stable linkage. TheEDC/sulfo-NHS treated β-purothionin is then bound to the amine end ofthe TFCS linker.

6.3.1 TFCS Treatment of CNT2 Polyclonal

A 1 ml aliquot Protein A purified rabbit 1 polyclonal IgG (see Example4.2.1, harvest bleed from rabbit 1 was affinity purified and thenpurified using a Protein A column) was concentrated using a centriconspin concentrator with 10 kDa cut off. The resulting sample volume wasup to 5 mg/ml 0.1M Sodium Phosphate buffer 0.15M NaCl pH 7.2. 15 ul TFCS(3 mg/ml in DMF) was added per 500 μl of antibody and incubated for 1hour at room temperature. The reaction mixture was applied to a PD10desalting column equilibrated with 0.1M phosphate buffer pH 8. All ofthe 3.5 ml sample volume was collected and concentrated using acentricon concentrator to obtain TFCS-treated anti-CNT2 antibody at afinal concentration of 10 mg/ml.

6.3.2 EDC and Sulfo-NHS Treatment of β-Purothionin

Lyophilised β-purothionin from wheat endosperm (Takara) was dissolved in0.1M Sodium Phosphate buffer 0.15M NaCl pH 7.2 to final concentration of10 mg/ml. EDC was added to give a final concentration 2 mM along withsulfo-NHS to a final concentration of 5 mM, and the mixture was left toreact at room temperature for 15 minutes. The reaction was stopped byadding 2-mercaptoethanol to a final concentration of 20 mM. Theactivated β-purothionin was applied to a polyacrylamide desalting column(size exclusion limit of 1,800 Da; Pierce) equilibrated with 0.1M SodiumPhosphate buffer, 0.15M NaCl pH 7.2. Fractions were collected and theOD280 nm checked for the presence of the toxin.

6.3.3 Conjugation of Anti-CNT2 Polyclonal Antibody to Activatedβ-Purothionin

TFCS-treated antibody and EDC/sulfo-NHS treated β-purothionin were mixedto give a 30:1 molar ratio of β-purothionin:anti-CNT2 antibody and leftto react at 4° C. overnight. The reaction was then stopped by addinghydroxylamine to a final concentration 10 mM. The reaction mixture wasthen applied to a size exclusion chromatography column to separate freeβ-purothionin from antibody-β-purothionin conjugate. Fractionscontaining the conjugate were pooled.

6.3.4 Analysis of Conjugate

MALDI-TOF mass-spectrometry was sued to analyse the composition of thepooled conjugate sample relative to unconjugated anti-CNT2 antibody. Themass spectra obtained (not shown) indicated the presence of 1:1, 1:2 and1:3 ratios of antibody-β-purothionin conjugate species in the pooledconjugate fractions. Some singly charged unconjugated antibody specieswas also observed.

The functional activity of the conjugate in the pooled sample wasassessed by ELISA as described previously. The 50% binding titre for theconjugate was calculated at 0.067 μg/ml in comparison to 0.041 μg/ml forunconjugated anti-CNT2 antibody.

TABLE 13 Immunohistochemical analysis of rat gastro-intestinal tissuewith anti-CNT2::β- purothionin conjugate Dilution of conjugate Blockedwith with Dilution staining Rat blocking range present Rat Duodenum RatJejunum Ileum Rat Colon peptide 1:25-1:50 1:25 & Muscularis Not NotMuscularis Not 1:50 mucosa tested tested mucosa tested staining andstaining villi and and villi crypt and crypt epithelial epithelial cellscells staining staining

A sample of the anti-CNT2::β-purothionin conjugate was used forimmunohistochemical analysis of rodent gastro-intestinal tissue (seeExample 4.2 for methodology). The results are summarised in Table 13above.

Example 7 Fusion Protein Expression Vector Construction

Fusion protein expression vectors were constructed between a scFvrecognising a cell-surface antigen and three different protein toxins,gelonin, granzyme B, and cyt2A. The scFvs used in this exemplificationare the SV63 scFvs described in Example 2.5 above. Standard molecularbiology techniques for plasmid preparation, restriction enzymedigestion, ligation, E. coli transformation etc were followedthroughout.

7.1 Gelonin-scFv Fusion Constructs

The gene encoding the 251 amino acid gelonin protein (see Nolan et al.supra) was synthesised artificially with the 5′ sequence designed topermit in-frame fusion to either the V_(H) (giving rise to artificialgene 054014) or V_(L) (giving rise to artificial gene 054013) encodingsequence from the SV63 antibody, and sub-cloned into pCR-Script to givethe two constructs p054013 and p054014.

Plasmid p054013 was digested with SpeI and NotI and the fragmentencoding gelonin that was generated was purified and ligated tosimilarly digested pDGF-SV63-VHVL, to give vector pDGF-SV63-VHVLrGel.This created an in-frame fusion between the N-terminus of the SV63 scFvvia a single Gly₄Ser linker to the recombinant Gelonin. Thus theexpression cassette in pDGF-SV63-VHVLrGel comprises the followingcomponents in the 5′ to 3′ direction: tac promoter, pelB leadersequence, SV63 V_(H) coding sequence, [Gly₄Ser]₃ linker, SV63 V_(L)coding sequence, Gly₄Ser linker, recombinant Gelonin encoding sequence.

The SV63 V_(H) coding sequence, [Gly₄Ser]₃ linker, SV63 V_(L) codingsequence, Gly₄Ser linker, and recombinant Gelonin encoding sequence, caneasily be excised as a NcoI/EcoRI fragment and ligated into alternativeexpression/cloning vectors, for example into pIMS147 or pET32a, ifdesired.

Plasmid p054014 was digested with BseRI and NotI and the fragmentgenerated encoding gelonin was purified and ligated into similarlydigested pDGF-SV63-VLVH, to give vector pDGF-SV63-VHVLrGel. This createdan in-frame fusion between N-terminus of the scFv via a single Gly₄Serlinker to the recombinant Gelonin. Thus the expression cassette inpDGF-SV63-VHVLrGel comprises the following components in the 5′ to 3′direction::tac promoter, pelB leader sequence, SV63 V_(L) codingsequence, [Gly₄Ser]₃ linker, SV63 V_(H) coding sequence, Gly₄Ser linker,recombinant Gelonin encoding sequence.

The SV63 V_(L) coding sequence, [Gly₄Ser]₃ linker, SV63 V_(H) codingsequence, Gly₄Ser linker, and recombinant Gelonin encoding sequence, caneasily be excised as a NcoI/EcoRI fragment and ligated into alternativeexpression/cloning vectors, for example into pIMS147 or pET32a, ifdesired.

7.2 Granzyme B-scFv Fusion Constructs

Two constructs were made, each carrying Granzyme B fused N-terminal to aSV63 scFv: pET32a::rGrzB::SV63 VHVL and pET32a::rGrzB::SV63 VLVH.

Plasmid pET32a::rGrzB::SV63 VHVL was constructed using pET32a(+)::rGrzB(Example 5.3 above) as a template in conjunction with a primer overlapextension approach to fuse mature GranzymeB coding sequence to the SV63scFv in the V_(H)V_(L) orientation (N to C-terminus) via a singleGly₄Ser linker.

(i) 3′ terminal sequence encoding a single Gly₄Ser linker was added tothe mature Granzyme B coding sequence by using pET32a(+)::rGrzB as atemplate for PCR with pET F (SEQ ID NO: 54) and RoPro 071 G4S/rGrzB R1(SEQ ID NO: 55) primers.

(ii) 5′ terminal sequence coding for a single Gly4Ser linker and part ofthe Granzyme B coding sequence was added to the SV63 VHVL codingsequence, using the pDGF-SV63-VHVL as a template for PCR with RoPro 074rGrzB/G4S/VHVL F (SEQ ID NO: 56) and RoPro 075 pET/VHVL R (SEQ ID NO:57) primers.

Products from (i) and (ii) above were purified and mixed in equimolaramounts prior to primer-free extension and subsequent PCR using pET F(SEQ ID NO: 54) and RoPro 075 pET/VHVL R (SEQ ID NO: 57) to amplify afull-length fusion product that was cloned into pET32a(+) using uniqueSfuI and NotI sites.

Plasmid pET32a::rGrzB::SV63 VLVH was also constructed usingpET32a(+)::rGrzB (Example 5.3 above) as a template in conjunction with aprimer overlap extension approach to fuse mature GranzymeB codingsequence to the SV63 scFv in the V_(L)V_(H) orientation (N toC-terminus) via a single Gly₄Ser linker.

(i) 3′ terminal sequence coding for a single Gly₄Ser linker was added tothe Granzyme B coding sequence as described in (i) above.

(ii) 5′ terminal sequence coding for a single Gly4Ser linker and part ofthe Granzyme B coding sequence was added to the SV63 VLVH codingsequence, using pDGF-SV63-VLVH as a template for PCR with RoPro 072rGrzB/G4S/VLVH F (SEQ ID NO: 58 and RoPro 073 VLVH/pET R (SEQ ID NO: 59)primers

Products from (i) and (ii) above were purified and mixed in equimolaramounts prior to primer-free extension and subsequent PCR using pET F(SEQ ID NO 54) and RoPro 073 VLVH/pET R (SEQ ID NO: 59) to amplify afull-length fusion product that was subsequently cloned into pET32a(+)using unique SfuI and NotI sites.

The sequences of the primers used in the construction of the GranzymeB-scFv fusion constructs are as described in Example 5.3 above and asfollows (all primers given 5′ to 3′): pET F (SEQ ID NO: 54)TCGGTGATGTCGGCGATATAG; RoPro 071 G4S/rGrzB R1 (SEQ ID NO: 55)ACTACCTCCGCCACCGGACTTCTTCATA GTTTTCTTGATCCAGG; RoPro 074 rGrzB/G4S/VHVLF (SEQ ID NO: 56) GAAGAAGTCCGGTGGCGGAGGTAGTGAGGTCCAGCTGCAGGAGTCTGGCCCTGG; RoPro 075 pET/VHVL R (SEQ ID NO: 57) TGCTCGAGTGCGGCCGCTTATTACTTGATCTCCAGTTTGGTGCCTCCACCGAACG; RoPro 072 rGrzB/G4S/VLVH F (SEQID NO: 58) GAAGAAGTCCGGTGGCGGAGGTAG TGATATCGTTCTCACTCAATCTCCAGCAATC;RoPro 073 VLVH/pET R (SEQ ID NO: 59)TGCTCGAGTGCGGCCGCTTATTATGAGGAGACTGTGAGAGTGGTG CCTTGGCC.

7.3 Cyt2A-scFv Fusion Constructs

In order to create in-frame fusions of the SV63 scFv and Cyt2A sequencesthe example of Gurkan & Ellar (2003 Protein Expr. Purif. 29(1):103-16)was followed. The gene encoding the first 237 amino acids of Cyt2Aa1(EMBL:BTCYTBG) was synthesised artificially to contain a 3′ sequencedesigned to create an in-frame C-terminal addition of the 14 amino acidXpress epitope (Invitrogen). This artificially synthesised Cyt2A-Xpressepitope sequence is known as 051072, and was sub-cloned in to pCR Scriptto give p051072. The plasmid pDGF-SV63-VHVL (see Example 2.5) wasaltered in two steps. Firstly, an artificially synthesised sequence wasdesigned and produced (named p054247) that would create a linkingfragment by containing part of the 3′ terminal sequence of the SV63 VLsequence, the (Gly₄Ser)₃ linker and part of the 5′ terminal sequence ofthe mature Cyt2Aa1 protein (as described by Gurkan & Ellar, supra). Thisfragment was excised from p054247 using SpeI (5′) and EcoRI (3′), andligated into similarly prepared pDGF-SV63-VHVL (see Example 2.5). Theresulting plasmid was then further modified by cutting with SfuI andBamHI and introducing the approximately 520 bp SfuI/BamHI fragment(representing the 3′ end of the mature Cyt2Aa1 sequence plus the Xpressepitope) from p051072. The resulting plasmid (pDGF-SV63-VHVL::Cyt2A)comprises a complete in-frame fusion of the SV63 V_(H)V_(L) scFv via aflexible linker to the active (mature) form of Cyt2Aa1 (amino acids 37to 237) followed by the Xpress epitope.

Similarly, the plasmid pDGF-SV63-VLVH was modified in a two-step processto introduce the Cyt2Aa1 sequence. Firstly, an artificially synthesisedsequence was designed and produced (named 054248) that would create alinking fragment by containing part of the 3′ terminal sequence of theSV63 VH sequence, the (Gly₄Ser)₃ linker and part of the 5′ terminalsequence of the mature Cyt2Aa1 protein (as described by Gurkan & Ellar,supra). The artificial sequence was sub-cloned into pCR Script to giveplasmid p05428. This fragment was then excised from p054248 using BseRI(5′) and EcoRI (3′), and ligated into similarly prepared pDGF-SV63-VLVH.The resulting plasmid was then further modified by cutting with SfuI andBamHI and introducing the approximately 520 bp SfuI/BamHI fragment(representing the 3′ end of the mature Cyt2Aa1 sequence plus the Xpressepitope) from p051072. The resulting plasmid (pDGF-SV63-VLVH::Cyt2A)comprises a complete in-frame fusion of the SV63 V_(L)V_(H) scFv via aflexible linker to the active (mature) form of Cyt2Aa1 (amino acids 37to 237) followed by the Xpress epitope

Example 8 Expression of Fusion Proteins

The vectors carrying the SV63 scFv-gelonin fusion genes (see Example 7.1above) were transformed into E. coli TOP10 cells. Pilot expressionstudies were performed by growing the transformed cells in 2×YT/2%glucose medium, before inducing at either 20° C. or 15° C. in 2×YTmedium supplemented with 1 mM IPTG for 16 hours. Small samples (10 mls)were harvested and centrifuged and each cell pellet was resuspended in 1ml lysis buffer (PBS). The samples were sonicated, re-centrifuged at13,000 rpm for 5 minutes at room temperature, and the supernatants(soluble fractions) decanted. The pellets (insoluble fractions) wereresuspended in 1 ml lysis buffer.

Soluble and insoluble fractions were analysed by Western blot using anantibody directed to the hexa-histidine tag. The analysis revealed thatscFv-gelonin fusion protein was being produced from both expressionvectors (i.e. where the scFv was in the V_(H)V_(L) orientation and wherethe scFv was in the V_(L)V_(H) orientation). However, the V_(L)V_(H)orientation of the scFv gave a higher yield of soluble protein.

Larger scale growth and induction was carried out, and scFv-geloninfusion protein was further purified from the soluble fraction throughpurification on a Gravitrap column.

Example 9 In Vitro Testing of Protein Conjugates and Fusion Proteins

The ability of protein conjugates and fusion proteins of the inventionto cause damage to the upper gastrointestinal tract is assessed bymeasuring the permeability of the mucosa to the non-absorbable marker,mannitol, using sections of isolated rat duodenum. Tissues isolated invitro are exposed to these rodent control agents, using a modificationof a method published previously (Heylings, 1991, Toxicol. Appl.Pharmacol. 107:482-493).

Briefly, a 10 cm section of gastrointestinal tract (immediately distalto the stomach) from adult male Alderley Park strain rats (Ap:Ak_(f)SD)is removed immediately after humane termination. The tissue is placed inoxygenated TC199 media and any food debris is carefully flushed out ofthe gut using TC199 media from the end furthest away from the stomach.Two sections of duodenum are prepared, each 2.5 cm in length, theproximal section (immediately after the stomach) and the distal section(immediately after entrance of the bile duct).

The sections are carefully attached taut by means of ligatures to theopen ends of two glass tubes connected to a reservoir. This allows theluminal (mucosal) and blood-side (serosal) surfaces of the isolatedmusosa to be bathed by separate solutions. The gap between the ends ofthe glass rods in each mucosal chamber is 12 mm. A diagrammaticrepresentation of the apparatus is similar to that shown in the upperpart FIG. 1 from Heylings 1991 supra. The chamber with mucosa attachedis rinsed with oxygenated TC199 media several times to remove excessmucus on the luminal side of the tissue. The luminal (mucosal) side isfilled with 4 ml of TC199 media containing 20 mg mannitol/ml (TC199-M),and the mucosa is checked for leakage. The mucosal chamber is immersedin an outer cup-shaped glass vessel filled with 40 ml TC199 media(serosal side solution) which is gassed with 95% O₂:5% CO₂. The chamberis positioned to avoid hydrostatic pressure gradients between the twobathing solutions. Both solutions are maintained at 37±0.1° C. by meansof a water jacket connected to an external pump. After a 10 minutepre-incubation period the mucosal chamber is removed and flushed throughwith TC199-M media to remove any mucus build up, finally the luminalside is filled with 4 ml of TC199-M containing ¹⁴C-Mannitol (anon-electrolyte that is poorly absorbed by the gastrointestinal tract)at a concentration of 5×10⁵ dpm/ml and returned to the glass chamber.

In order to measure the permeability of the isolated duodenal mucosa tomannitol, duplicate 150 μl aliquots of the serosal side solution aretaken at 10, 20, 30, 60, 120, 180 and 240 minutes, following itsaddition to the mucosal side. The amount of mannitol absorbed isdetermined by liquid scintillation counting. As a positive control,paraquat (40 mg paraquat ion/ml), a known topical irritant to thegastrointestinal tract, is added to the mucosal chamber 30 minutes afterthe start of the incubation in order to demonstrate that the model iscapable of detecting mucosal damage. The rodent control agents (fusionproteins or protein conjugates, see for example Examples 6 to 8 above)are added by direct addition of a small volume to the mucosal chamber at30 minutes after the start of the incubation and the time course profileof mannitol absorption is monitored. This is compared with contemporarynegative controls for both proximal and distal segments of rat duodenumrun in parallel.

The methodology described above uses duodenal tissue, however, otherareas of the gastrointestinal tract may be substituted and tested usingthe same procedure as described above.

Example 10 In Vivo Testing of Protein Conjugates and Fusion Proteins10.1 Testing for Efficacy by Oral Gavage in Mouse

Mice (18 in total, 9 per group) will be dosed orally by gavage with i)protein conjugate or fusion protein (see in particular Examples 6 to 8above; group 1 mice), or ii) inert vehicle (e.g. polyethylene glycol;group 2 mice). Animals will be terminated 24, 48 and 72 hours postdosing, with clinical observations being made regularly throughout thestudy. Following termination, duodenal, jejunal, ileal and colonictissue will be fixed in formal buffered saline, processed and embeddedinto wax and stained with H&E for pathological assessment.

Fusion proteins/protein conjugates are employed at the following testconcentrations (given in mg of compound per kg bodyweight): 8 mg/kg, 5mg/kg and 3 mg/kg.

1. A rodent control agent comprising an antibody component that binds toan extracellular epitope of a protein that is expressed in a rodent. 2.A rodent control agent according to claim 1 that is adapted to killrodents or prevent breeding in rodents.
 3. A rodent control agentaccording to claim 1, wherein the antibody component is linked to atoxic component or a contraceptive component.
 4. A rodent control agentaccording to claim 3 a fusion protein, said fusion protein having afirst protein component and a second protein component, said firstprotein component being the antibody component and said second proteincomponent being selected from the group consisting of a toxin, animmunogen and a hormone.
 5. A rodent control agent according to claim 4,wherein said first and second protein components are linked to eachother via a peptide linker.
 6. A rodent control agent according to claim5, said peptide linker includes Glycine and Serine residues.
 7. A rodentcontrol agent according to claim 6, wherein said peptide linker includesat least three (Gly₄Ser) motifs.
 8. A rodent control agent according toclaim 1 including a protein conjugate chemically conjugated to a toxiccomponent or a contraceptive component.
 9. A rodent control agentaccording to claim 3, wherein the toxic component is a protein toxin.10. A rodent control agent according to claim 9, wherein said toxin isa) a full-length protein selected from the group of proteins listed ingroup 1, wherein group 1 consists of a protein which disrupts membranes,a ribosyltransferase, a serine protease, a guanylyl cyclase activator, aprotein involved in ATPase mediated ion transport, acalmodulin-dependent adenylyl cyclase and a ribonuclease, or is b) atoxic domain of a protein selected from group 1
 11. A rodent controlagent according to claim 9, wherein said toxin is a) a full-length RNAglycosidase or is b) a toxic domain of a RNA glycosidase.
 12. A rodentcontrol agent according to claim 10, wherein said toxin is a)full-length β-purothionin or a full-length protein selected from thegroup of proteins listed in group 2, wherein group 2 consists ofPerfingolysin O, Alpha-haemolysin, Sphingomelinase, Delta-haemolysin,Granzyme B, Alpha toxin, Cyt toxin, Diphtheria toxin, Granulysin,Melittin, Perforin, Cholera enterotoxin, Heat-stable enterotoxin,Equinatoxin, Listeriolysin, VIP2, Accessory enterotoxin, Aerolysin,BinA, BinB, Colicin E1, Haemolysin A, CTX IV, Ricin, Amoebapore, El Torhaemolysin, Vibrio damsela haemolysin, Pneumolysin, Streptolysin O,Kanagawa toxin, Leptospira haemolysin, Cry toxin, Anthrax toxin,Pseudomonas exotoxin A, Barnase, and VIP3, or is b) a toxic domain ofb-purothionin or a toxic domain of a protein selected from group
 2. 13.A rodent control agent according to claim 11, wherein the toxin isgelonin.
 14. A rodent control agent according to claim 3 wherein thecontraceptive component is an immunogen capable of eliciting a responseagainst an ovum or sperm-specific antigen.
 15. A rodent control agentaccording to claim 3, wherein the contraceptive component is areproductive hormone.
 16. A rodent control agent according to claim 15,wherein the reproductive hormone is gonadotrophin releasing hormone. 17.A rodent control agent according to claim 8, wherein the toxic componentis a toxic compound selected from the group consisting of: colchicine;doxorubicin; calicheamicin; a non-steroidal anti-inflammatory drug(NSAID) compound; cytochalasin; an anticoagulant; calciferol;bromethalin; flupropadine; zinc phosphide; scilliroside; sodium(mono)fluoroacetate; fluoroacetamide; alphachloralose; thalliumsulphate.
 18. A rodent control agent according to claim 17, wherein theanti-coagulant is selected from the group consisting of: brodificoum,difenacoum, bromadiolone, flocoumafen, difethialone, hydroxycoumarins,and indane-diones.
 19. A rodent control agent according to claim 8,wherein the contraceptive component is a hormone or hormone-likecompound.
 20. A rodent control agent according to claim 19, wherein thehormone-like compound is diazacon.
 21. A rodent control agent accordingto claim 1, wherein the protein to which the antibody component binds isa protein expressed in the gastro-intestinal epithelium of a rodent. 22.A rodent control agent according to claim 21, wherein the protein isselected from the group consisting of Rat PEP T1, Rat CD155, Rat GTR2,Rat CFTR, Rat CNT2, Rat CATB(0+), Rat MDR1, Mouse MDR1, RatSucrase-Isomaltase, Mouse GLUT7, Rat GTR5, Rat Npt2A, Rat OAT-B, RatASBT, Rat CAT1, Rat OATP3, Rat ABCG8, Rat GTR8, Rat MRP1, Rat CNT1, RatUT-B, Rat DRA1, Mouse ENT1 and Rat ENT1.
 23. A rodent control agentaccording to claim 21, wherein the protein is selected from the groupconsisting of Rat CATB(0+), Rat GCC, Rat PLB, Rat LPH, Mouse LPH, RatAMPN, Rat MCDL, Rat SCAB, Rat KCV2.
 24. A rodent control agent accordingto claim 22, wherein the extracellular epitope is provided by a an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-26.25. A rodent control agent according to claim 24, wherein theextracellular epitope is provided by an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4 and SEQ ID NO:
 5. 26. A rodent control agent according toclaim 23, wherein the extracellular epitope is provided by an amino acidsequence selected from the group consisting of SEQ ID NOs: 27-36.
 27. Arodent control agent according to claim 1, wherein the antibodycomponent is an antibody or an antigen binding fragment thereof.
 28. Arodent control agent according to claim 27 wherein the antibody ispolyclonal.
 29. A rodent control agent according to claim 27 wherein theantibody is monoclonal.
 30. A rodent control agent according to claim 27wherein the antibody is a) an immunoglobulin comprising light and heavychains or b) a single chain antibody.
 31. A rodent control agentaccording to claim 30, wherein the single-chain antibody is a scFv. 32.A rodent control agent according to claim 30, wherein the single chainantibody is devoid of light chains.
 33. A rodent control agent accordingto claim 32, wherein the single-chain antibody is derived from theCamelidae or from the Chondrichthyes.
 34. A rodent control agentaccording to claim 1, wherein the antibody component is selected fromthe group consisting of an immunoglobulin light chain, an immunoglobulinheavy chain, a V_(H) domain, a V_(L) domain, Fv, Fab, di-Fab, Fab′,F(ab′)₂, a VHH domain, an IgNAR V domain and a CDR.
 35. A rodent controlagent according to claim 1, wherein the antibody component is disulphidestabilised.
 36. A rodent control agent according to claim 1, wherein theantibody component binds to a rodent protein with greater affinity thanto a homologous protein from at least one non-target animal.
 37. Arodent control agent according to claim 36, wherein the non-targetanimal is selected from the group consisting of humans, birds, companionanimal, farm animals and wild animals that are not pests.
 38. A rodentcontrol agent according to claim 37, wherein the non-target animal ishuman.
 39. A rodent control agent according to claim 36, wherein theantibody component exhibits displaceable binding to the extracellularepitope of the rodent protein, but does not exhibit displaceable bindingto: i) a homologous protein from a non-target animal, or; ii) acorresponding epitope from the homologous protein from a non-targetanimal.
 40. A rodent control agent according to claim 39 wherein theextracellular epitope of the rodent protein is represented by a rodentspecific peptide epitope consisting of an oligopeptide fragment of aprotein expressed in a rodent, wherein the oligopeptide fragmentsequence represents an extracellular continuous peptide epitope that hasa percentage identity of 60% or less with a corresponding linear peptidesequence from a homologous protein from a non-target animal.
 41. Arodent control agent according to claim 1, to wherein the protein thatis expressed in a rodent is an essential protein.
 42. A rodent controlagent according to claim 41 wherein the essential protein is expressedin the gastrointestinal epithelium of a rodent.
 43. A rodent controlagent comprising an antibody component linked to a protein toxin,wherein the antibody component binds to an extracellular epitope of aprotein that is expressed in a rodent and wherein the affinity ofbinding of the antibody component to the extracellular epitope of therodent protein is greater than the affinity of binding of the antibodycomponent to a homologous protein from a non-target animal.
 44. A rodentcontrol agent according to claim 1 in the form of a compositionincluding at least one additive.
 45. A rodent control agent according toclaim 44 wherein said additive is a first generation anti-coagulant or asecond generation anti-coagulant.
 46. A rodent control agent accordingto claim 44, wherein at least one additive has the function of makingthe composition palatable to rodents.
 47. A method of killing a rodent,comprising: (A) providing a rodent control agent including an antibodycomponent that binds to an extracellular epitope of a protein that isexpressed in the rodent, said rodent control agent adapted to kill therodent upon ingestion thereof; and (B) placing said rodent control agentin an area frequented by the rodent.
 48. A method of preventing rodentsfrom breeding, comprising: (A) providing a rodent control agent,including an antibody component that binds to an extracellular epitopeof a protein that is expressed in the rodent, said rodent control agentadapted to impair the reproductive capabilities of said rodent uponingestion thereof; and (B) placing said rodent control agent in an areafrequented by the rodent.
 49. A rodent specific peptide epitope (RSPE)consisting of an oligopeptide fragment of a protein expressed in arodent, wherein the oligopeptide fragment sequence represents anextracellular continuous peptide epitope that has a percentage identityof 60% or less with a corresponding linear peptide sequence from ahomologous protein from a non-target animal.
 50. An antibody, or anantigen-binding fragment thereof, which binds to the RSPE of claim 48.51. (canceled)